Method and apparatus for screening obscured or partially obscured cells

ABSTRACT

A method and apparatus for performing screening of obscured or partially obscured cells to enumerate one or more cell population subsets. For example, a whole blood sample or portion thereof is screened to provide the desired analysis of one or more white blood cell population subsets in the sample. A white blood cell population including the obscured subset of interest is first counted, along with a standard population. The standard population can be one of the total number of white blood cell populations, a second white blood cell population which does not obscure the shifted or non-shifted sensed characteristic of the subset of interest, an artificial population formed by microspheres which also do not obscure the shifted or non-shifted sensed characteristic of the subset of interest or a white blood cell population into which the sensed characteristic of the subset will be wholly or partially shifted. The sensed characteristic of the white blood cell population subset of interest then is shifted by binding microspheres having monoclonal antibodies specific to the white blood cell population subset of interest to the cell population. The white blood cell population, and the standard population then are again counted and compared to the original counts to obtain an enumeration of the white blood cell population subset of interest.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of applications, U.S. Ser.No. 07/339,156, filed Apr. 14, 1989, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 09/285,856, filed Dec. 16, 1988,now abandoned which in turn is a continuation-in-part of U.S. Ser. No.07/025,345, filed Mar. 13, 1987, now abandoned in favor of continuationapplication U.S. Ser. No. 587,646, now U.S. Pat. No. 5,223,398 filedSep. 20, 1990, the disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

This invention relates generally to a method and apparatus for screeningcells which are obscured or are partially obscured by another cellpopulation, which cells express selected characteristics for research,diagnostic or industrial purposes. More particularly, the invention isdirected to an analysis of the obscured cells by utilizing microsphereshaving specific monoclonal antibodies bound thereto to move the sensedcharacteristics of the obscured cells from one cell population toanother.

This invention relates generally to an automated analyzer and methods ofusing same for screening biological cells or formed bodies for theenumeration of populations which express selected characteristics forresearch, diagnostic, medical or industrial purposes. More particularly,the automated analyzers and methods embodying the invention enablemultiple part classifications of cells and formed bodies, functionalphenotyping of cells and formed bodies, typing of leukemic, lymphoma andsolid tumor cells, among others, using a unique combination ofelectronic and optical technology and the specificity of selectivebiological molecules, such as antibodies, for such screening andselective enumeration of the cells and formed bodies.

Automation of routine complete blood cell (CBC) analysis of humanperipheral blood by an automated blood cell counter was successfullyachieved by the COULTER COUNTERS@ Model A of Coulter Electronics, Inc.of Hialeah, Fla. The electronic particle sensing system principle ofthat instrument is disclosed in U.S. Pat. No. 2,656,508 issued Oct. 20,1953 to Wallace H. Coulter. The use of optical sensing means or lasers,which can be troublesome and expensive are avoided by particle analyzinginstrumentation solely operated on this Coulter electronic sensingprinciple.

This Coulter sensing principle was developed and expanded into moresophisticated instrumentation such as the COULTER COUNTER@ Model S typesof instruments which enabled CBC parameters, absolute cell counts,platelet count and morphology, red blood cell (RBC) morphology,interpretation of normal and abnormal blood specimens by specificcomputer programs.

The Coulter electronic particle sensing principle employs an aperturesensing circuit using a direct current (DC) aperture supply. Suchparticle sensors are simple in structure, extremely rugged and reliableas attested to by the substantially universal acceptance of the COULTERCOUNTER@ automated analyzer in clinical laboratories in the UnitedStates and throughout the rest of the World. An improvement in thisbasic aperture sensing circuit was disclosed in U.S. Pat. No. 3,502,974issued in 1970 to Wallace Coulter and Walter Hogg. In addition to thestandard direct current aperture supply, a high frequency aperturecurrent was applied which enabled the sensing of an additional parameterfor classification purposes. The high frequency aperture currentproduced a signal which is the function of the blood cell's internalconductivity as well as its volume. The signal produced simultaneouslyby the direct current aperture circuit is a conventional DC amplitudesignal which provides an indication primarily of cell volume. The radiofrequency amplitude is divided by the direct current pulse amplitudeemploying a high speed divider circuit to obtain a quotient which is afunction of cell volume and internal resistance, conveniently referredto as "opacity". This principle is further described in U.S. Pat. No.3,502,973 also issued to Wallace Coulter and Walter Hogg, in 1970. Thisparameter has applicability in cell classification systems. Either asingle or a pair of separate apertures could be utilized for thispurpose.

Classification of different populations is accomplished by collating thedata of the signal pairs as they are produced; one, a measure ofparticle volume and the other a measure of cell internal resistivity oropacity. A convenient form of presenting this data is by two-dimensionalplots referred to as scatterplots or scattergrams. Such plots are welldescribed in Flow Cytometry and Sorting, page 371; edited by MelamedMelaney, and Medelsohn, 1979, John Wiley & Sons, New York, N.Y.

FIG. 5A is one example of a data plot of a sample of normal blood. Eachdot represents an individual cell. The height above the baselinerepresents the relative volume of the cell. The distance of the dot tothe right of the vertical baseline represents the relative opacity. Aplot of normal white blood cells (WBC) (with the red blood cellsremoved) shows three clusters of dots representing three distinctpopulations which are a consequence of their intrinsic differences insize and internal composition. If desired, with suitable circuitry,these populations can be enumerated to obtain the numbers of each. Thecells are classified on the basis of these inherent differences.

Initial applications of the Coulter electronic particle sensingprinciple was to perform red blood cell counts and then, moresophisticated determinations of other red blood cell parameters. Byremoving red blood cells from whole peripheral blood, analysis of thewhite blood cell populations could be undertaken so long as the redblood cell removal did not significantly impair properties of theremaining white blood cell populations sought to be measured. Red bloodcell lysing reagents were developed for this purpose which, thoughuseful and widely applied, were not entirely satisfactory in allrespects for subsequent white blood cell determinations.

Previous methods of flow analysis of leukocytes using DC volume alone orlight scatter at various angles have shown three clusters of leukocytescorresponding to lymphocytes, monocytes and granulocytes which includedthe neutrophil, basophil and eosinophil populations. A rough but usefulestimation of eosinophil concentration can be made on some samples. Thefifth major population is relatively too small for this approach. Theeosinophils also have been observed as a distinct cluster using specialfluorescence techniques.

These fluorescent techniques were utilized in flow cytometry instrumentssuch as the EPICS@ flow cytometer available from the CoulterCorporation. Such instruments employed the principle of cells moving ina columnar stream bounded by a sheath flow such that cells lined up insingle file and passed individually through a laser beam. Light scatterand/or fluorescence signals from the cells were then utilized inclassifying cell populations. Staining cells with absorptive orfluorescent dyes made additional cell population classificationspossible. The development of instrumentation and fluorochromes forautomated multiparameter analysis is further described by R. C. Leif, etal. in Clinical Chemistry, Vol. 23, pp. 1492-98 (1977). Thesedevelopments expanded the number of simultaneous populationclassifications of leukocytes to four, namely lymphocytes, monocytes,eosinophils and "granulocytes" (neutrophils and basophils).

A more recent analytical hematology instrument has utilized lightscattering techniques together with peroxidase enzyme staining(absorptive dye) of cells to produce a five part leukocyte differential.Moreover, dyes in combination with specific reacting biologicalmolecules, such as monoclonal antibodies, have increased the number ofleukocyte classifications possible to include functional subdivisions.

An improved single automated instrument and methods of using the same isdisclosed in a first parent application, U.S. Ser. No. 025,345, filedMar. 13, 1987, now abandoned in favor of continuation application U.S.Ser. No. 587,646, now U.S. Pat. No. 5,223,398 filed Sep. 20, 1990,entitled AUTOMATED ANALYZER AND METHOD FOR SCREENING CELLS OR FORMEDBODIES FOR ENUMERATION OF POPULATIONS EXPRESSING SELECTEDCHARACTERISTICS. This parent application combines the application ofelectronic sensing aperture principles, the specificity of selectedbiological molecules for identifying and/or enumerating definedpopulations of cells or formed bodies and microscopic particletechnology. The automated analyzer can be used together with a speciallysing reagent and/or antibodies coupled to microscopic microspheres orsupports of varying composition.

A second parent application, U.S. Ser. No. 285,856, filed Dec. 16, 1988,entitled METHOD AND APPARATUS FOR SCREENING CELLS OR FORMED BODIES WITHPOPULATIONS EXPRESSING SELECTED CHARACTERISTICS, discloses the screeningof direct subsets from whole blood samples or portions thereof.

A third parent application, U.S. Ser. No. 339,156, filed Apr. 14, 1989,entitled METHOD AND APPARATUS FOR SCREENING CELLS OR FORMED BODIES WITHPOPULATIONS EXPRESSING SELECTED CHARACTERISTICS UTILIZING AT LEAST ONESENSING PARAMETER discloses multipart or five part white blood celldifferentials, lymphocyte subsets and overlapping determinationsperformed from a whole blood sample or from a sample with the red bloodcells and/or populations of the white blood cells removed by eliminationof populations and/or subsets thereof with one or more light orelectronic parameters. Each of the parent and co-pending applicationsreferred to herein now are commonly assigned.

Selectively attaching microscopic particles makes possible themodification of the parameter(s) responsible for the original locationof at least one of the populations. The bulk addition of microscopicparticles to selected target populations where this addition affects themeasured volume and/or opacity results in shifting the location of thedots representing a population.

Antibodies of known specificity are employed in coating microscopicparticles. This coating gives the particle the capacity to selectivelyattach to certain cells which express the antigen the antibody isspecific for. These coated or tagged cells are a combination ofparticles and cell which behave like a new entity. Their parameters ofopacity, volume, or both opacity and volume may be considered torepresent the sum of the effects of both the cell and the particles onthe signals obtained. If the characteristics of the components aredifferent, the new entity will move to a new position in accordance withthe net effect. The new location, in contrast with the former positionof the cell alone, should allow a classification of such new entity orgroup of new entities. If the particles attached to the cells aremagnetic, then, of course, according to current practice, the newentities can be captured by the use of a magnet. If mixed rapidly,unexpected results including complete capture of a population withoutadversely affecting the properties of the cells under study occur.

Only three distinct populations of cells can be readily identified andenumerated from a blood sample by utilizing their inherent and uniqueproperties of DC volume and opacity parameters heretofore stated.Additional steps such as improved lysing systems, must be taken toenable the detection and enumeration of more populations. Of course,these additional populations represent subpopulations of the three basicones referred to as lymphocytes, monocytes and granulocytes. The stepsperformed in accordance with the parent application demonstrate howsubpopulations of these basic three populations are obtained.

Employing such simple aperture sensing techniques in combination withtwo or more biological particles, one can produce a unique and newposition of the dot cluster representing a given population. Thisselective movement of populations on the dot plot or scattergram isreproducible and can be used to classify a population separate from thebasic three populations.

The original and inherent combination of DC volume and opacity sensingtechniques can be modified through the attachment of microscopicparticles to selected individual cells. The selectivity is given theparticles by the nature or specificity of the biological molecules,antibodies among others, employed as the coating on their surfaces. Apopulation of cells alone, having no particles on their surface, mayoccupy a dot plot position no different from other populations orsubpopulations, and, henceforth, not be distinguishable from oneanother. The addition of particles having a selective attraction to aspecific population of cells which one seeks to identify, enumerate, andstudy is possible using this approach. The selective addition of asufficient mass of selective particles to a distinct population ofinterest results in the shifting of that population's dot plot locationas a result of the new and unique combination of mass, volume andopacity.

The separation of specific cell populations is accomplished withoutmaterially affecting the properties of remaining cell populations. Forexample, the removal of erythrocytes or red blood cells (RBC's) fromwhole blood in accordance with this invention permits the measurement ofT4 and/or T8 lymphocytes not otherwise possible with heretoforeavailable chemical RBC lysing reagents. Ratios of the number of T4versus T8 cells have been used to indicate immune deficienciesconsistent with severe viral infections including the AIDS virus amongothers. The presence of specific receptors on the surface of cells canbe used to classify a population into subsets, whose enumeration permitsthe detection of the onset of disease. For example, in the predominantforms of leukemia there is a sharp rise in peripheral blood lymphocytes.If the subpopulation of lymphocytes which is rapidly proliferating bearsthe T11 receptor, the patient is at risk of immune abnormalities.

Further, if the subpopulation of T1 positive lymphocytes is T4 receptorbearing, then the patient is classified as that leukemia common inJapan. These cells are defined as "overlapping" since the cells includeat least two receptors or antigens of interest. Overlapping can be asignificant parameter in diagnosis and treatment. An example ofoverlapping populations in a normal whole blood sample is the CD2 andCD8 subset populations. Another example of an abnormal overlapping ofpopulations is found in CLL (chronic lymphocytic leukemia). In the CLLdisease state, the CD5 and CD20 subset populations overlap. Moreover, ifthe T4 receptor subpopulations expanding is 2H4 positive, then thepatient will not only demonstrate a tendency of multiple infections butacute leukemia as well for the T11, T4, 2H4 positive cell is the inducerof suppression and functionally inhibits the patient's ability to makeantibodies. Therein the patient is subject to multiple infections andmust be treated for both leukemia and immune deficiency. K. Takatsuki,et al., GANN monograph on Cancer Research 28:13-22, 1982; C. Morimoto,et al., Coulter Japan Symposium, 1984; C. Morimoto, et al., Immunology134 (3):1508-1515, 1985; C. Morimoto, et al., New England Journal ofMedicine 316(2):67-71, 1987. The invention also applies to analyses offormed body suspensions such as bacteria and viruses among others.

The method and apparatus embodying the invention can be utilized with avariety of immunological reactions, such as immunological reactionsinvolving reacting agents and formed bodies or cells. The invention alsoapplies to analyses of formed body suspensions such as some bacteria andviruses among others. As utilized herein, cells are defined as animal orplant cells, including cellular bacteria, fungi, which are identifiableseparately or in aggregates. Cells are the least structural aggregate ofliving matter capable of functioning as an independent unit. Forexample, cells can be human RBC and WBC populations, cancer or otherabnormal cells from tissue or from blood samples. Formed bodies aredefined as some bacteria and viruses. The invention can be utilized indiagnosing and monitoring patients. The invention specifically can beutilized to eliminate or shift populations to analyze populations orsubpopulations which cannot otherwise easily be identified. The cellsand formed bodies suitably tagged or labeled reasonably can be expectedto be sensed by the method and apparatus of the invention in the samemanner as the human blood cell examples.

Although the term "reactant" has been utilized in the parent applicationto define lysing agents and monoclonal antibodies, reactants can includevarious agents which detect and react with one or more specificmolecules which are on the surface of a cell or formed body. Someexamples are given below:

    ______________________________________                                        Reactant          Specific Molecule                                           ______________________________________                                        Antibody          Antigen                                                     Drug              Drug Receptor                                               Hormone           Hormone Receptor                                            Growth Factor     Growth Factor Receptor                                      ______________________________________                                    

The reactants couple or bind to the specific molecule(s) on the cells.These reactants do form part of a chemical reaction; however, thereactants are not necessarily chemically altered.

This invention provides a single versatile analyzer and methods of usingsame which combines a minimum of electronic and/or light particlesensing technology and the specificity of selective biological moleculesto enable a major advancement in the field of automated analyzers forclinical laboratory use, and for industrial applications. The detectionof multiple leukocyte subpopulations, and their relationship to oneanother in human peripheral blood is important in medical research andthe diagnosis of human diseases. Such data are useful as a screeningtool for identifying and classifying diseases, such as leukemia.Abnormal situations identified by implementation of the invention hereinprovides diagnostically relevant information in areas of study notlimited only to detection of leukocyte populations as will be apparentfrom the specification and drawings hereof.

One of the most valuable features of this invention is that it employsthe single rugged Coulter sensing operation. It is stable and does notrequire the complexity and expense of complex optical systems but canutilize light sensing if desired. The circuitry required for theaddition of the RF generator and detector is economical, compact andreliable. A single aperture is all that is required, but the addition ofa second or even a third aperture can enable a greater sample throughputrate economically.

SUMMARY OF THE INVENTION

The invention provides a method and apparatus for performing screeningof obscured or partially obscured cells to enumerate one or moreobscured cells such as white blood cell population subsets. The obscuredor white blood cell population subset is enumerated by identifyingselected characteristics expressed by the cells of interest.

A whole blood sample or portion thereof can be screened to provide thedesired analysis of one or more obscured cells such as white blood cellpopulation subsets in the sample. For example purposes, the white bloodcell population is described in detail. A white blood cell populationincluding the obscured subset of interest is first counted, along with astandard population. The standard population can be one of the totalnumber of white blood cell populations, a second white blood cellpopulation which does not obscure the shifted or non-shifted sensedcharacteristic of the subset of interest, an artificial populationformed by microspheres which also do not obscure the shifted ornon-shifted sensed characteristic of the subset of interest or a whiteblood cell population into which the sensed characteristic of the subsetwill be wholly or partially shifted. The sensed characteristic of thewhite blood cell population subset of interest then is shifted bybinding microspheres having monoclonal antibodies specific to the whiteblood cell population subset to the cell population. The white bloodcell population, and the standard population then again are counted andcompared to the original counts to obtain an enumeration of the whiteblood cell population subset of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-13 describe the embodiments disclosed in first parentapplication Ser. No. 025,345;

FIG. 1 is a schematic block diagram of one cell population analyzerembodiment of the parent application;

FIG. 2 is a schematic block diagram of a second analyzer embodiment ofthe parent application;

FIG. 3 is one specific analyzer embodiment of the parent applicationcorresponding to FIGS. 1 and 2;

FIG. 4 is a schematic block diagram of another analyzer embodiment ofthe parent application;

FIGS. 5A and 5B are a scattergram of one set of results utilizing aprototype analyzer system similar to that illustrated with respect toFIGS. 2 and 3;

FIG. 6 is a schematic block diagram of a further analyzer embodiment ofthe parent application;

FIG. 7 is a schematic block diagram of a still further analyzerembodiment of the parent application;

FIGS. 8A and 8B, 9A and 9B, 10A and 10B and, 11A and 11B are ascattergram of one set of results utilizing a prototype analyzer systemsimilar to that illustrated with respect to FIGS. 6 and 7;

FIG. 12 is a schematic block diagram of a yet still further analyzerembodiment of the parent application;

FIG. 13 is a scattergram of one set of results utilizing a prototypeanalyzer system similar to that illustrated with respect to FIG. 12;

FIGS. 14-26D describe the embodiments disclosed in the second parentapplication Ser. No. 285,856;

FIG. 14 is a schematic block diagram of one WBC population subsetanalyzer embodiment of the parent application;

FIG. 15 is another schematic block diagram of a WBC population subsetanalyzer embodiment of the parent application;

FIG. 16 is one specific analyzer embodiment of the parent applicationcorresponding to FIGS. 14 and 15;

FIGS. 17A and 17B are a scattergram of one set of results utilizing aprototype analyzer system similar to that illustrated with respect toFIGS. 3 and 16;

FIG. 18A is a scattergram of the L, M and G populations and FIG. 18B isa scattergram of the L, M and B populations utilizing a prototypeanalyzer system similar to that illustrated with respect to FIG. 16;

FIGS. 19A-D, 20A-D and 21A-D are scattergrams of the CD4, CD8, CD2 andCD20 subset populations of samples of different patients;

FIG. 22A is a scattergram similar to the scattergram of FIG. 18A,

FIG. 22B is a scattergram illustrating shifting of the E and Npopulations and

FIG. 22C is a scattergram illustrating shifting of the E, N and CD4populations;

FIGS. 23A-D are scattergrams illustrating a direct WBC subset analysisutilizing one microsphere bound to the WBC subset of interest and asecond microsphere bound to the first microsphere;

FIGS. 24A-C are scattergrams illustrating the effect of the size of themicrosphere utilized in the shifting analysis of the parent application;

FIGS. 25A-D are scattergrams illustrating a simultaneous analysis of twoWBC subset populations by the techniques of the parent application;

FIGS. 26A-D are scattergrams of the same populations illustrated ondifferent parameter scattergrams;

FIGS. 27-46 describe the embodiments of the third parent applicationSer. No. 339,156;

FIG. 27 is a schematic block diagram of one single sensing parametermultipart WBC population subset analyzer embodiment of the parentapplication;

FIG. 28 is one specific analyzer embodiment of the parent applicationcorresponding to FIG. 27;

FIGS. 29A-D are scattergrams of one set of results utilizing a prototypeanalyzer system and a DC sensing parameter similar to that illustratedwith respect to FIGS. 27 and 28;

FIGS. 30A-D are scattergrams of the same results of FIGS. 29A-Dutilizing an RF sensing parameter;

FIGS. 31A-D are scattergrams of a second set of results utilizing theprototype analyzer system and a DC sensing parameter similar to thatillustrated with respect to FIGS. 27 and 28.

FIGS. 32A-D are scattergrams of the same results of FIGS. 31A-Dutilizing a RF sensing parameter;

FIGS. 33-36 are scattergrams of results illustrating the use of a singlelight sensitive parameter;

FIG. 37 is a schematic block diagram of a WBC population subset analysisfor enhancing small or obscure populations;

FIGS. 38A-E are scattergrams of one set of results obtained utilizing aprototype analyzer system and an RF sensing parameter similar to thatillustrated with respect to FIGS. 27 and 37;

FIGS. 39A-E are scattergrams of another set of results obtainedutilizing a DC sensing parameter;

FIGS. 40A-E are scattergrams of the same set of results utilizing an RFsensing parameter;

FIGS. 41A-E are scattergrams of one set of results obtained utilizingtwo sensing parameters;

FIGS. 42A-G and 43A-G are scattergrams of results obtained utilizingvarious sensing parameters on two respective abnormal blood samples;

FIG. 44 is a schematic block diagram of a WBC subset populationanalyzing embodiment of the invention for determining overlappingclassification of cells;

FIGS. 45A-D and 46A-D are scattergrams of two sets of results utilizinga prototype analyzer system and a DC sensing parameter similar to thatillustrated in FIGS. 27 and 44;

FIGS. 47-60 are directed to embodiments of the present invention;

FIG. 47 is a schematic block diagram of one white blood cell populationsubset analyzer of the present invention;

FIGS. 48A and B are conceptual scattergrams illustrating the methodologyutilized in the present invention; and

FIGS. 49-60 are scattergrams of results utilizing the methodology of thepresent invention to analyze obscured or partially obscured white bloodcell population subsets.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIGS. 1-13 describe the embodiments of the first parent application,Ser. No. 025,345.

Referring to FIG. 1, a first embodiment of a cell population analyzingmethod and apparatus of the parent application, Ser. No. 025,345, isdesignated generally by the reference numeral 10. The analyzer 10includes a biological sample 12 which contains at least a first set ofviable biological cells (not illustrated), such as in or from a wholeblood sample. The cells of the biological sample 12 are to be involvedin a biological reaction in a quantitative and/or qualitativedetermination or analysis. The sample 12 can include a buffer into whichthe cells are added.

The sample 12 is combined via a line 14 with at least one reactant 16via a line 18. The red blood cells (RBC) then are removed from themixture by a functionally designated RBC removing station 20. The RBC'scan be removed from the mixture by the station 20 in a number of ways.The RBC's can be lysed by a lyse in the reactant 16. One suchpreferential lyse and a quench which can be utilized therewith isdisclosed in Ser. No. 130,911, filed Dec. 10, 1987, now abandoned infavor of continuation application U.S. Ser. No. 611,378, filed Nov. 13,1990, now abandoned entitled METHOD AND REAGENT SYSTEM FOR ISOLATION,IDENTIFICATION AND/OR ANALYSIS OF LEUKOCYTES FROM WHOLE BLOOD SAMPLES,which is a CIP of Ser. No. 025,303, filed Mar. 13, 1987, now abandonedin favor of continuation application U.S. Ser. No. 317,147, filed Feb.28, 1989, now abandoned of the same title, which are incorporated hereinby reference. The reactant 16 can be or include a plurality of magneticmicrospheres with an antibody specific to the RBC's bound to themicrospheres (not illustrated). In this example, the particular redblood cell specific antibody utilized is disclosed in application Ser.No. 799,489, filed Nov. 19, 1985, now U.S. Pat. No. 4,752,563, entitledMONOCLONAL ANTIBODY FOR RECOVERY OF LEUKOCYTES IN HUMAN PERIPHERAL BLOODAND METHOD OF RECOVERY EMPLOYING SAID MONOCLONAL ANTIBODY, which isincorporated herein by reference. The reactant 16 also can include abuffer in addition to or in place of the sample buffer. The reactant 16further can be a combination of the preferential RBC lyse and the RBCspecific microspheres.

Once the RBC's substantially are removed from the mixture, a portion ofthe mixture is fed into a white blood cell (WBC) analyzer 22 via a line24. The WBC analyzer 22 at least counts the number of WBC's in themixture. The WBC analyzer 22 also can measure one or more volume oropacity parameters of the WBC's. The results from the analyzer 22 arefed to a comparator 26 via a line 28.

A second portion of the RBC deleted mixture is fed to a WBC subsetsubtracting station 30 via a line 32. The WBC's can be subtracted fromthe mixture in a number of ways. Microspheres with a monoclonal antibodyspecific to one of the WBC subsets bound thereto can be added to themixture. Non-magnetic microspheres can be bound to the WBC's to changeor shift the resultant opacity or volume parameters of the cells.Magnetic microspheres also can be bound to the WBC's which then can beremoved from the mixture by a magnetic field.

The mixture with the WBC subset population removed or with one or moreparameters changed then is fed to a WBC subset analyzer 34 via a line36. The analyzer 34 can be identical to the analyzer 22. The results ofthe analyzer 34 are then fed to the comparator 26 via a line 38. Thecomparator 26 then can compare the WBC results from the analyzer 22 withthe modified results from the analyzer 34 to determine at least onecharacteristic of the selected white blood cell population, such as thenumber of cells in a particular range.

Referring to FIG. 2, a second embodiment of a cell population analyzingmethod and apparatus embodying the parent application is designatedgenerally by the reference numeral 40. The analyzer 40 includes abiological sample 42 which again contains at least a first set of viablebiological cells (not illustrated), such as in or from a whole bloodsample. The cells of the biological sample 42 are to be involved in abiological reaction in a quantitative and/or qualitative determinationor analysis. The sample 42 again can include a buffer into which thecells are added.

The sample 42 is combined via a line 44 with at least one reactant 46via a line 48. In the analyzer 40, the RBC's are removed from themixture and simultaneously at least one characteristic of at least oneWBC subset is changed or shifted by a functionally designated RBCremoving and WBC shifting station 50. As stated above, the RBC's can beremoved from the mixture by the station in a number of ways, previouslyenumerated with respect to the station 20. Simultaneously, in the samemixture portion, the WBC's are bound to, generally non-magnetic,microspheres to change or shift the resultant opacity and/or volumeparameters of the cells.

The mixture with the RBC's removed and the WBC subset population shiftedthen is fed to an analyzer 52 via a line 54. The analyzer 52 can besubstantially identical to the analyzer 22. The analyzer 40 thusprovides a fast, direct analysis of at least one characteristic of aselected WBC population or WBC subset.

One specific embodiment of an analyzer instrument embodying the parentapplication and which can accomplish the analyzing methods of the firstand second analyzers 10 and 40, is designated generally by the referencenumeral 56 in FIG. 3.

In the instrument 56, only one specific enumeration is illustrated,which can be varied in almost endless detail in accordance with theprinciples of the parent application. Further, the instrument 56 isshown in generally functional detail and the specific embodiments can bestructurally implemented in many known ways.

The instrument 56 includes an aspirator pumping mechanism 58 which isutilized to draw the biological sample of interest, for example thesample 12 or 42 into the instrument 56. The aspirator 58 is coupled viaa line 60 to a sampling valve 62, which can be coupled to a sample probe63. A lyse pump 64 can include the lyse, such as part of the reactant 18or 46 and is also coupled to the valve 62 via a line 66. The valve 62and the pump 58 can aspirate the biological sample 12 or 42 along withthe lyse via the pump 64 when appropriate.

The reactant mixture or the biological sample itself, then is fed via adischarge line 68 into a mixing apparatus 70. The mixer 70 includes amixing chamber 72 into which the sample or reactant is fed. At thispoint the operation of the analyzer 10 and 40 differ and hence will bedescribed separately.

In the case of the analyzer 10, if the RBC's have been lysed by the lysefrom the pump 64, then when the reaction is completed a quench or fix issupplied from a station 74 via a line 76. The reaction can be assistedby mixing the lyse and the sample in the chamber 72 as illustratedfunctionally at 78.

Specific details of an appropriate mixing apparatus 70, which can beutilized herein are disclosed in Ser. No. 025,337, filed Mar. 13, 1987,now abandoned in favor of continuation application U.S. Ser. No.517,309, filed May 1, 1990, now U.S. Pat. No. 5,238,812 entitled METHODAND APPARATUS FOR RAPID MIXING OF SMALL VOLUMES FOR ENHANCING BIOLOGICALREACTIONS, which is incorporated herein by reference. By utilizing themixer 70 the reactions are greatly enhanced in speed withoutsignificantly damaging the properties of interest of the cells, such as,can occur by raising the reaction temperature. Further, the reactionsgenerally are completed in significantly less than a minute, generallyon the order of fifteen seconds or less. This allows a rapid analysis ofthe automatic high volume analyzer instrument 56.

The quenched reactant with the RBC's removed by the lyse (as from thestation 20) then is fed via a line 80 to a holding chamber 82, which inthis case will hold a second portion of the mixture. A first portion ofthe mixture will be fed from the chamber 82 via a line 84 to a WBCanalyzer 86 (i.e., analyzer 22). The analyzer 86 can be of many physicaltypes in accordance with the counting and sizing techniques described byWallace H. Coulter in U.S. Pat. No. 2,656,508 and embodied in thenumerous commercial blood cell counters of the assignee, CoulterElectronics, Inc.

The analyzer 86, in general, includes a flow sensor or sensing chamber88. The chamber 88 includes a transducer 90 which has an aperture 92therethrough. The chamber 88 includes a first portion 94 which has afirst electrode 96 in contact with the fluid therein.

The chamber portion 94 and the electrode 96 communicate through theaperture 92 with a second chamber portion 98 having a second electrode100 therein. The electrodes 96 and 100 are coupled via reactive leads102 and 104 to an RF/DC source and sensing circuit 106. The circuit 106couples both a DC, or low frequency current or signal, and a highfrequency signal between the electrodes 96 and 100.

The low frequency signal is utilized to sense the amplitude of a signalpulse caused by a cell passing through the aperture 92. The highfrequency signal is utilized to obtain the electrical opacity of thesame cell passing through the aperture 92.

The measuring of the electrical opacity of cells was described byWallace H. Coulter and Walter R. Hogg in U.S. Pat. No. 3,502,974 andseveral patents and publications of the assignee, Coulter Electronics,Inc., since that patent. One specific circuit which can be utilizedherein is disclosed in Case 168,008, entitled PARTICLE ANALYZER FORMEASURING THE RESISTANCE AND REACTANCE OF A PARTICLE, filed Oct. 21,1986, now U.S. Ser. No. 921,654, now U.S. Pat. No. 4,791,355, which isincorporated herein by reference.

The signals generated by the circuit 106 from the sensed cells arecoupled via a DC signal lead 108 and an RF signal lead 110 to acomparator 112 (like the comparator 26). The comparator 112 can hold thesignal generated from the first portion, i.e., those without the WBCsubset subtracted, for a comparison with the results from the secondportion to be described.

The analyzer 86 can include a sheath flow to focus the cells in thesensor 88, in the well known manner. The sheath flow can be provided bya fluidic system 114, coupled to the sensor 88 by a pair of lines 116and 118 in a known manner. The sample reaction mixture can be fed intothe sensor 88 via an introduction tube 120 and can be fed from thesensor 88 via an exit tube 122 into a waste container 124.

While the first portion of the mixture was being analyzed in theanalyzer 86, the second portion is held in the chamber 82, while themixer 72 is cleaned or flushed via a rinse line 126 and exhaustedthrough a waste line 128. Once the chamber 72 is cleaned, the secondportion is fed back into the chamber 72 via a line 130. Like the station30, the WBC subset now is subtracted by adding the WBC microspheres froma station 132 via a line 134, a valve 136 and a chamber line 138.

The WBC microspheres are mixed with the second portion by the mixingmechanism 78. If the WBC microspheres are non-magnetic, the reactionmixture with the bound WBC microspheres is fed via the line 80, thechamber 82 and the line 84 into the analyzer 86 (i.e., the analyzer 34),wherein the second portion is analyzed like the first portion and theresults then are compared in the comparator 112 (i.e., the comparator26). At least one of the WBC subset cell parameters is changed in thesecond portion, such as the cell opacity by the WBC subset boundmicrospheres to provide the changed results which then can be analyzed.

If the WBC microspheres are magnetic, then the WBC subset bound theretoare removed by a magnetic field during and/or after the mixing processby a magnetic field or magnet 140. The field can be provided byelectromagnetic means or by the magnet 140 being physically moved withrespect to the chamber 72 to capture the magnetically bound WBC subset.The second portion without the bound WBC subset then is fed via the line80, the chamber 82 and line 84 to the analyzer 86 in the mannerpreviously described to obtain the analysis (like the analyzer 34).

The instrument 56 then is prepared to take the next sample for the nextanalysis. The probe 63 can be cleaned by a probe rinse mechanism 142 andthe lines and chamber 72 and 82 can be flushed in a conventional manner.Each analysis of the succeeding sample mixture is obtained in a rapidand automatic fashion. The period between the analysis of succeedingsample mixtures can be on the order of minutes or less.

In operating the analyzer instrument 56, like the analyzer 40, thereaction mixture with the RBC lyse/reactant 46 and the sample 42 ismixed in the chamber 72 along with non-magnetic WBC microspheres fromthe station 132, which bind to one of the WBC subsets. The quench 74 isadded to the reactive mixture which then is fed via the line 80, thechamber 82 and the line 84 to the WBC analyzer 86 for analysis (i.e.,like the analyzer 52).

Alternatively to the utilization of the lyse, in either of the analyzers10 and 40, the sample 12 or 42 can be fed to the mixer 70 via the valve62 without any lyse. In this case, the RBC's can be removed magneticallyby utilizing the microspheres with the RBC specific antibody boundthereto from an RBC microsphere station 144 and fed to the valve 136 viaa line 146 and hence to the chamber 70 via the line 138. Where no lyseis utilized, the bound RBC's are magnetically removed by the magnet 140after mixing in a manner substantially identical to the magneticallybound WBC's described above.

Further, in a second case to promote the speed of the reaction, areaction mixture of the sample with both the RBC lyse and with the RBCmagnetic beads can be utilized. The reaction mixture is mixed, the lyseis quenched and the bound RBC's are magnetically removed and then theWBC's are analyzed as previously described.

Referring now to FIG. 4, another embodiment of a cell populationanalyzing method and apparatus embodying the parent application isdesignated generally by the reference numeral 148. The analyzer 148includes a biological sample 150 which again contains at least a firstset of viable biological cells, such as in or from a whole blood sample.The sample 150 again can include a buffer into which the cells areadded.

The sample 150 is combined via a line 152 with at least one reactant 154via a line 156. The RBC's then are removed as above described by afunctionally designated RBC removing station 158. The reaction mixturewith the RBC's removed is fed via a line 160 into a WBC analyzer 162.The results from the analyzer 162 are fed to a comparator 164 via a line166, providing a three-part WBC differential with results for monocytes(M), lymphocytes (L) and granulocytes (G).

The mixture then is fed to a neutrophil (N) functionally designatedremoval station 168 via a line 170. The N's can be removed from themixture by shifting or changing one parameter, such as opacity, or bymagnetic removal, both as described above. In this example, theparticular N specific antibody utilized is disclosed is Case 168,306,MONOCLONAL ANTIBODY SPECIFIC TO NEUTROPHILS, filed Dec. 8, 1986, nowU.S. Ser. No. 938,864, now abandoned in favor of continuation-in-partapplication U.S. Ser. No. 070,202, filed Jul. 6, 1987, now U.S. Pat. No.4,931,395.

The mixture with the N's removed or shifted then is fed to another WBCanalyzer 172 via a line 174. The results of the analyzer 172 are fed tothe comparator 164 via a line 176. The results of the analyzer 172 areutilized to obtain a four-part WBC differential with results again forM's and L's, but now in addition since the N's are shifted or removedresults for eosinophils (E) and basophils (B) are obtained. The twoanalytical results from the analyzers 162 and 172 then can be comparedby the comparator 164 to form a five-part WBC differential.Specifically, subtracting the number of B's and E's from the number ofG's results in the number of the removed N's.

Referring now to FIGS. 5A and 5B, two sets of scattergram results areillustrated obtained from a whole blood sample utilizing a prototypeanalyzing method similar to the analyzer 148. The biological sample 150was a 20 microliter sample of whole blood which was combined with 40microliters of the magnetic microspheres with the RBC specific antibodybound thereto combined with 140 microliters of buffer solution to formthe reactant 154. The reaction mixture was mixed for 15 seconds andplaced in a magnetic field for 10 seconds in the station 158. Themixture with the RBC's removed was analyzed by the analyzer 162 asillustrated in the scattergram of FIG. 5A resulting in counts of L's of45.6 (1), M's of 5.6 (2) and G's of 48.7 (3).

The mixture then is combined in the station 168 with 10 microliters ofmagnetic microspheres with the N specific antibody bound thereto. Themixture is mixed 30 seconds and then placed in a magnetic field for 10seconds. The mixture with the N's then removed was fed to the analyzer176 which resulted in the scattergram of FIG. 5B resulting in counts ofL's of 81.0 (1), M's of 0.6 (2), E's of 11.0 (3) and B's of 1.8 (4). Thecomparator 164 then provides the five-part WBC differential of counts of45.6 L's, 5.6 M's, 41.6 N's, 6.0 E's and 1.2 B's. This corresponds to astandard microscopic five-part WBC differential utilizing Wright stainon the sample on a slide resulting in counts of 44.0 L's, 3.4 M's, 45.0N's, 6.1 E's and 0.4 B's.

FIG. 6 illustrates a further embodiment of a cell population analyzingmethod and apparatus embodying the parent application, designatedgenerally by the reference numeral 178. The analyzer 178 includes abiological sample 180 which again contains at least a first set ofviable biological cells and also can include a buffer.

The sample 180 is combined via a line 182 with a reactant 184 via a line186. Functionally illustrated, a first portion of the mixture is fed viaa line 188 to a functionally designated RBC and N removing station 190.The RBC's and N's are removed or shifted as described before and thefirst portion is fed via a line 192 to a WBC analyzer 194.

This provides a result from the analyzer 194 which is fed via a line 196to a comparator 198. The results includes the above-referenced four-partdifferential including M's, L's, E's and B's.

At the same time, a second portion of the mixture of the sample 180 andthe reactant 184 is fed via a line 200 to a functionally designated RBCremoval station 202. The mixture with the RBC's removed is fed via aline 204 to another WBC analyzer 206. The results of the analyzer 206are fed to the comparator 198 via a line 208. The results of theanalyzer 206 directly include the above-referenced three-part WBCdifferential including M's, L's and G's. the results of the analyzers194 and 206 then are compared by the comparator 198 to provide thefive-part WBC differential.

A specific analyzing instrument embodiment incorporating the method andapparatus of the analyzer 178 is designated generally by the referencenumeral 210 in FIG. 7. Again, only one specific hardware enumeration hasbeen illustrated, but like the analyzing instrument 56, the analyzinginstrument 210 can be implemented in numerous configurations.

The instrument 210 includes an aspirator purging mechanism 212 which iscoupled to a sampling valve 214 via a line 216. The valve 214 caninclude a sample probe 218 to aspirate the biological sample ofinterest, such as the sample 180. A diluent delivery pump 220 is coupledto the valve 214 via a line 222 to provide a diluent for the sample,such as a whole blood sample, when desired. A first portion of themixture then is coupled via a line 224 and a line 226 to a first mixingapparatus 228. At the same time, a second portion of the mixture is fedvia the line 224 and a line 230 to a second mixing apparatus 232.

The mixer 228 (comparable to the station 190) is substantially identicalto the mixer 232 (comparable to the station 202) and will be describedfirst. The mixer 228 includes a mixing chamber 234 into which the firstmixture portion is fed. The mixer 228 includes all of the variousoptions above described and can include a lyse input line 236 for theRBC lyse if desired.

If the lyse is utilized, after mixing as illustrated functionally at238, then the quench is added via a quench line 240. At the same time,the N's are being removed by the addition of the appropriate magnetic ornon-magnetic microspheres with the N specific antibody bound theretofrom a source of microspheres 242 fed to the chamber 234 via a line 244.If magnetic microspheres are utilized for the N's or the RBC's, then amagnet 246 or magnetic field is utilized to remove the magneticallybound cells.

The mixed and quenched (where necessary) mixture then is fed via a line248 through a valve 250 and a line 252 to a WBC analyzer 254 (i.e.,analyzer 194). The analyzer 254 is the same as the analyzer 86 and willnot be described again in such detail. Again, the analyzer 254 includesa sensing chamber 256 with an aperture 258 therein through which themixture and cells pass. A sheath flow fluidic system 260 can be coupledto the chamber 256. The signals generated by the cells are detected byan RF/DC source and sensing circuit 262 whose outputs are fed to acomparator 264, as previously described.

Concurrently, the second mixture portion is fed into a mixing chamber266. In the second portion, only the RBC's are removed (i.e., like thestation 202) and the RBC's can be removed by the RBC lyse fed into thechamber 266 via a line 268. The lyse is mixed with the sample and then aquench is added via a quench line 270. Alternatively the RBC's can beremoved by magnetic microspheres having the RBC specific antibody boundthereto from a microsphere source 272 fed into the chamber 266 via aline 274. The microspheres are mixed, functionally at 276, and then themagnetically bound RBC microspheres are removed by a magnet 278.

The RBC removed mixture then is fed via a line 280 to the valve 250 andvia the line 252 to the analyzer 254 to obtain the above-mentionedresults. The mixers 228 and 232 include appropriate respective rinselines 282 and 284 and waste lines 286 and 288 and a probe rinse 290 tocleanse the instrument 210 prior to aspirating the next sample or samplefor analyzing.

FIGS. 8A and 8B illustrate scattergram results obtained from a wholeblood sample utilizing an analyzing method similar to the analyzer 178.In this example, 20 microliters of whole blood form the sample 180,while 40 microliters of magnetic microspheres with the RBC specificantibody bound thereto combined with 140 microliters of buffer solutionform the reactant 184. A portion of the mixture is mixed for 20 secondsin the station 202 and then placed in a magnetic field for 10 seconds.The RBC removed mixture then is analyzed in the analyzer 206 resultingin the scattergram of FIG. 8A which provides a count of L's 29.4 (1),M's 8.1 (2) and G's 62.4 (3).

At the same time, another portion of the same mixture is combined with10 microliters of magnetic microspheres with the N specific antibodybound thereto to remove the RBC's and N's in the station 190. Themixture is mixed for 30 seconds, then placed in a magnetic field for 10seconds. The mixture with the N's and RBC's removed then is analyzed bythe analyzer 194 resulting in the scattergram of FIG. 8B which providesa count of L's 73.5 (1), M's 21.7 (2), E's 3.4 (3) and B's 1.4 (4). Thetwo counts are compared in the comparator 198, resulting in a five-partWBC differential count of L's 29.4, M's 8.0, N's 60.8, E's 1.2 and B's0.6. A microscope comparison again was made resulting in counts of L's29.4, M's 5.0, N's 65.0, E's 1.0 and B's of less than 1.0.

FIGS. 9A and 9B show scattergram results of a five-part WBC differentialexample similar to that of FIGS. 8A and 8B. A 20 microliter sample ofwhole blood was analyzed in the same steps described with respect toFIGS. 8A and 8B resulting in the scattergram of FIG. 9A providing acount of L's 35.4 (1), M's 14.6 (2) and G's 50.0 (3). The scattergram ofFIG. 9B provides a count of L's 66.4 (1), M's 25.0 (2), E's 6.6 (3) andB's 2.0 (4). The resulting five-part WBC differential results in countsof 35.4 L's, 14.6 M's, 45.5 N's, 3.5 E's and 1.1 B's was compared to amicroscope count of 36 L's, 11 M's, 49 N's, 3 E's and 1 B.

FIGS. 10A and 10B show scattergram results of a five-part WBCdifferential again similar to that of FIGS. 8A, 8B and 9A, 9B, however,in this example, lyse was utilized. In this example, 20 microliters ofwhole blood was combined with 80 microliters of buffer and 240microliters of the RBC preferential lyse above referenced. The mixtureis mixed for 6 seconds and then a quench is added. The time period issignificant, because the lyse left unquenched for a period of timegreater than about 10 seconds will start to affect the significantproperties of the WBC's. The mixture with the RBC's removed is analyzedto provide the scattergram of FIG. 10A resulting in counts of L's 25.7(1), M's 9.6 (2) and G's 65.0 (3).

A second portion of the mixture including a second 20 microliter sampleof the whole blood is combined with 120 microliters of buffer and 10microliters of magnetic microspheres with the N specific antibody boundthereto and mixed for 30 seconds and then placed in a magnetic field for10 seconds. The RBC preferential lyse then is added to the N removedmixture which then is mixed for 6 seconds before it is quenched. Theresulting scattergram FIG. 10B results in percentage counts of L's 74.6(1), M's 21.6 (2), E's 2.9 (3) and B's 0.8 (4). The resulting five-partWBC differential results in percentage counts of L's 25.6, M's 9.6, N's63.5, E's 1.06 and B's 0.3. Again a microscope comparison resulted incounts of L's 29.4, M's 5.0, N's 65.0, E's 1.0 and B's of less than 1.

Another example of scattergram results of a five-part WBC differentialsimilar to that of FIGS. 10A and 10B is illustrated in FIGS. 11A and11B. A sample of whole blood had two samples simultaneously analyzed inthe same steps described with a respect to FIGS. 10A and 10B. Thescattergram of FIG. 11A provides a count of L's 31.9 (1), M's 17.6 (2)and G's 50.4 (3). The scattergram of FIG. 11B provides a count of L's67.1 (1), M's 24.1 (2), E's 7.6 (3) and B's 1.2 (4). The resultingfive-part WBC differential results in counts of 31.9 L's, 11.4 M's, 46.0N's, 3.6 E's and 0.7 B's as compared to a microscope count of 36 L's, 11M's, 49 N's, 3 E's and 1 B's.

A yet still further embodiment of a cell population analyzing method andapparatus embodying the parent application is designated generally bythe reference numeral 292 in FIG. 12. The analyzer 292 includes abiological sample 294, again including at least a first set of viablebiological cells and including a buffer if desired.

The sample 294 is combined via a line 296 with at least one reactant 298via a line 300. In the analyzer 292, the RBC's are removed and the N'sare shifted sequentially or simultaneously in a functionally designatedstation 302. The RBC remove function is designated 304 and the N move orshift portion is designated 306 to indicate that the functions can beperformed simultaneously or sequentially. The RBC's can be removedmagnetically or with lyse or with a combination of the two as previouslydescribed. The N's are removed or shifted by adding microspheres havingan N specific antibody bound thereto to the mixture.

Once the RBC's are removed and the N's are moved or shifted, then theresulting mixture is fed via a line 308 to an analyzer 310. In thiscase, the N's are shifted sufficiently from the patterns of the E's andB's that a five-part WBC differential of M's, L's, E's, B's and N's isdirectly obtained. The functions of the analyzer 292 can be performed oneither of the instruments 56 and 210 or minor variations thereof.

The scattergram results of one example of a direct five-part WBCdifferential in accordance with the analyzer 292 is illustrated in FIG.13. In this example, the biological sample 294 is 20 microliters of awhole blood sample and the reactant 298 is 10 microliters ofnon-magnetic microspheres with the N specific antibody bound theretocombined with 100 microliters of buffer and mixed in the substation 306for 30 seconds. The RBC preferential lyse, 10 microliters thereof, thenis added to the mixture which is mixed in the substation 304 for 6seconds after which the quench is added. The RBC removed and N shiftedmixture then is analyzed by the analyzer 310 resulting in thescattergram of FIG. 13 which provides a direct count of 29.6 L's, 13.6M's, 52.2 N's, 3.4 E's and 1.06 B's as compared to a microscopedetermination of 35 L's, 5 M's, 56 N's, 4 E's and no B's. In thisparticular example, the whole blood sample was also analyzed on ageneral cell counting instrument of Coulter Electronics, Inc., whichresulted in 29 L's, 11.1 M's and 59.9 G's (N's, E's and B's).

Referring now to FIGS. 14-26D, the embodiments of the second parentapplication Ser. No. 285,856 are illustrated.

Referring to FIG. 14, a first embodiment of a WBC population subsetanalyzer method and apparatus of the second parent application isdesignated generally by the reference numeral 320. The analyzer 320includes a biological sample 322, which contains at least a first set ofviable biological cells (not illustrated), including at least one whiteblood cell population having at least one definable subset, such as inor from a whole blood sample. As utilized herein, WBC subsets aresubsets of a WBC population to which specific monoclonal antibodies canbe bound. A nomenclature now has been defined for the monoclonalantibodies by the World Health Organization and the InternationalImmunology Society. The monoclonal antibodies are defined by a clusterof differentiation (CD) nomenclature which defines a particularspecificity for a cell or group of cells and the monoclonal antibodiesspecific for that CD group. For example purposes only, four CD groupshave been utilized in the following examples, CD4, CD8, CD2 and CD20.The CD nomenclature, specificity and some commercial sources ofmonoclonal antibodies are illustrated in Table I.

                  TABLE I                                                         ______________________________________                                        Cluster of                                                                              Antibody                                                            Differentiation                                                                         (Commercial Source).sup.b                                                                       Specificity                                       ______________________________________                                        CD2(gp 50).sup.a                                                                        T11 (Coulter)     E Rossette                                                  OKT11 (Ortho); Leu5.sup.a (BD)                                                                  Receptor                                          CD4(gp 56)                                                                              T4 (Coulter)      Helper/inducer                                    T         OKT4.sup.a (Ortho); Leu3.sup.a (BD)                                 CD8(gp 32-33)                                                                           T8 (Coulter)      Cytotoxic/                                                  OKT8 (Ortho); Leu2.sup.a (BD)                                                                   Suppressor T                                      CD20(gp 35)                                                                             B1 (Coulter)      All B cells ex-                                             Leu 16 (BD)       cept for plasma                                                               cells, B cell                                                                 tumors, except                                                                for myeloma,                                                                  some non-T ALL                                                                cells                                             ______________________________________                                         .sup.a gp  glycoprotein, molecular weight in kilodaltons                      .sup.b Coulter  Coulter Immunology Division of Coulter Corporation            (Hialeah, Florida)                                                            BD  BectonDickinson Immunocytometry Systems                                   Ortho  Ortho Diagnostic Systems (Raritan, New Jersey)                    

The cells of the biological sample 322 are to be involved in abiological reaction in a quantitative and/or qualitative determinationor analysis. The sample 322 can include a buffer into which the cellsare added.

The sample 322 is combined via a line 324 with at least one reactant 326via a line 328. In the analyzer 320, the RBC's are removed from themixture and simultaneously or sequentially at least one characteristicof at least one WBC subset is changed or shifted by a functionallydesignated RBC removing and WBC subset shifting station 330. As statedin the first parent application, the RBC's can be removed from themixture by the station 330 in a number of ways, such as enumerated withrespect to the station 20. Simultaneously or sequentially, in the samemixture portion, at least one WBC subset is bound to WBC microsphereshaving monoclonal antibodies specific to the subset thereon to modify(change or shift) the resultant opacity and/or volume parameters of thecells.

The mixture with the RBC's removed and the WBC subset populationshifted, then is fed to an analyzer 332 via a line 34. The analyzer 332can be substantially identical to the analyzer 22. The WBC subset ofinterest generally is related as a percentage of the WBC population ofinterest. The analyzer 320 thus provides a fast direct analysis of atleast one characteristic of a selected subset of a WBC population. Theanalyzer 320 can be utilized where the shifted WBC subset is notobscured by other more numerous cells, or where the number of theshifted cells of the WBC subset is a sufficient percentage as to beidentifiable, even though obscured.

Referring to FIG. 15, a second embodiment of a WBC population subsetanalyzing method and apparatus of the second parent application isdesignated generally by the reference numeral 340. The analyzer 340includes a biological sample, which contains at least a first set ofviable biological cells (not illustrated), including at least one whiteblood cell population having at least one subset, such as in or from awhole blood sample. The cells of the biological sample 342 again are tobe involved in a biological reaction in a quantitative and/orqualitative determination or analysis. The sample 342 can include abuffer into which the cells are added.

The sample 342 is combined via a line 344 with at least one reactant 346via a line 348. In the analyzer 340, the RBC's are removed from themixture and simultaneously or sequentially at least one characteristicof at least one WBC subset is changed or shifted by a functionallydesignated RBC removing and WBC subset shifting station 350. Aspreviously stated, the RBC's can be removed from the mixture by thestation 350 in a number of ways, such as enumerated with respect to thestation 20. Again, simultaneously or sequentially, in the same mixtureportion, at least one WBC subset is bound to microspheres to modify(change or shift) the resultant opacity and/or volume parameters of thecells.

At the same time or sequentially, at least one WBC population or subsetis removed from the mixture. The WBC population or subset is removed sothat the WBC subset of interest is not obscured by the population. Thispreferably is accomplished by magnetically removing the WBC populationafter they are bound to magnetic microspheres which include a monoclonalantibody bound thereto which is specific to the WBC population.

The mixture with the RBC's and the WBC population removed and the WBCsubset populations shifted then is fed to an analyzer 352 via a line354. The analyzer 352 again can be substantially identical to theanalyzer 22.

One specific embodiment of an analyzer instrument embodying the secondparent application and which can accomplish the analyzing methods of thefirst and second analyzers 320 and 340, is designated generally by thereference numeral 360 in FIG. 16.

In the instrument 360, like the instrument 56, only one specificenumeration is illustrated, which can be varied in almost endless detailin accordance with the principles of the first parent application.Further, the instrument 360 is shown in generally functional detail andthe specific embodiments can be structurally implemented in many knownways.

The instrument 360 includes an aspirator pumping mechanism 362 which isutilized to draw the biological sample of interest, for example thesample 322 or 342 into the instrument 360. The aspirator 362 is coupledvia a line 364 to a sampling valve 366 which can be coupled to a sampleprobe 368. A lyse pump 370 can include the lyse, such as part of thereactant 326 or 346 and is also coupled to the valve 366 via a line 372.The valve 366 and the pump 362 can aspirate the biological sample 322 or342 along with the lyse via the pump 370 when appropriate. Preferably,the biological sample 322 or 342 is added separately from the lyse.

The reactant mixture or the biological sample itself, then is fed via adischarge line 374 into a mixing apparatus 376. The mixer 376 includes amixing chamber 378 into which the sample or reactant is fed. Theanalyzers 320 and 340 differ only slightly in operation and hence willbe described together.

In operation, if the RBC's have been lysed by the lyse from the pump370, then when the reaction is completed a quench or fix is suppliedfrom a station 380 via a line 382. The RBC removal reaction then iscompleted. The reaction can be assisted by mixing the lyse and thesample in the chamber 378 as illustrated functionally at 384.

Either before, after or concurrently with the removal of the RBC's theWBC's are shifted and in the case of the analyzer 340, one WBCpopulation or subset also is removed. The WBC subset is shifted byadding the specific WBC microspheres from a station 386 via a line 388,a valve 390 and a chamber line 392. The WBC microspheres are mixed withthe mixture or the sample by the mixing mechanism 384.

The details of an appropriate mixing apparatus 376 can be substantiallyidentical to the mixing apparatus 70. By utilizing the mixer 376 thereactions are greatly enhanced in speed without significantly damagingthe properties of interest of the cells, such as, can occur by raisingthe reaction temperature. Further, the reactions generally are completedin significantly less than a few minutes and generally can be on theorder of two minutes or less. This allows a rapid analysis of theautomatic high volume analyzer instrument 360.

In the analyzer 320, the quenched reactant with the RBC's removed by thelyse (as from the station 20) and the modified WBC subset then is fedvia a line 394 to a WBC analyzer 396 (i.e., analyzer 332). The analyzer396 can be of many physical types in accordance with the counting andsizing techniques described by Wallace H. Coulter in U.S. Pat. No.2,656,508 and embodied in the numerous commercial blood cell counters ofthe assignee, Coulter Electronics, Inc.

As previously described, the analyzer 396, in general, includes a flowsensor or sensing chamber 398. The chamber 398 includes a transducer 400which has an aperture 402 therethrough. The chamber 398 includes a firstportion 404 which has a first electrode 406 in contact with the fluidtherein.

The chamber portion 404 and the electrode 406 communicate through theaperture 402 with a second chamber portion 408 having a second electrode410 therein. The electrodes 406 and 410 are coupled via reactive leads412 and 414 to an RF/DC source and sensing circuit 416. The circuit 416couples both a DC, or low frequency current or signal, and a highfrequency signal between the electrodes 406 and 410.

The low frequency signal is utilized to sense the amplitude of a signalpulse caused by a cell passing through the aperture 402. The highfrequency signal is utilized to obtain the electrical opacity of thesame cell passing through the aperture 402.

The measuring of the electrical opacity of cells was described byWallace H. Coulter and Walter R. Hoff in U.S. Pat. No. 3,502,974 andseveral patents and publications of the assignee, Coulter Electronics,Inc., since that patent. One specific circuit which can be utilizedherein is disclosed in U.S. Ser. No. 921,654, incorporated herein byreference.

The signals generated by the circuit 416 from the sensed cells arecoupled via a DC signal lead 418 and an RF signal lead 420 to acomparator 422 (like the comparator 26).

The analyzer 396 can include a sheath flow to focus the cells in thesensor 398, in the well known manner. The sheath flow can be provided bya fluidic system 424, coupled to the sensor 398 by a pair of lines 426and 428 in a known manner. The sample reaction mixture can be fed intothe sensor 398 via an introduction tube 430 and can be fed from thesensor 398 via an exit tube 432 into a waste container 434.

Following each operation, the mixer 378 is cleaned or flushed via arinse line 436 and exhausted through a waste line 438. Once the chamber378 is cleaned, another sample or sample portion can be fed into theinstrument 360.

In the analyzer 340, the operation is the same as the analyzer 320 withthe addition of magnetic white blood cell population or subsetmicrospheres. The WBC subset bound thereto then are removed by amagnetic field during and/or after the mixing process by a magneticfield or magnet 440. The field can be provided by electromagnetic meansor by the magnet 440 being physically moved with respect to the chamber378 to capture the magnetically bound WBC subset. The mixture withoutthe bound WBC subset then is fed via the line 394 to the analyzer 396 inthe manner previously described to obtain the analysis (like theanalyzer 320).

The instrument 360 then is prepared to take the next sample for the nextanalysis. The probe 368 can be cleaned by a probe rinse mechanism 442and the lines and chamber 378 can be flushed in a conventional manner.Each analysis of the succeeding sample mixture is obtained in a rapidand automatic fashion. The period between the analysis of succeedingsample mixtures can be on the order of five minutes or less.

Alternatively to the utilization of the lyse, in either of the analyzers320 and 340, the sample 322 or 342 can be fed to the mixer 376 via thevalve 366 without any lyse. In this case the RBC's can be removedmagnetically by utilizing microspheres with the RBC specific antibodybound thereto from an RBC microsphere station 444 and fed to the valve390 via a line 446 and hence to the chamber 376 via the line 392. Whereno lyse is utilized, the bound RBC's also are magnetically removed bythe magnet 440 after mixing in a manner substantially identical to themagnetically bound WBC's described above.

Further, in a second case to promote the speed or efficiency of thereaction, a reaction mixture of the sample with both the RBC lyse andwith the RBC magnetic beads can be utilized. The reaction mixture ismixed, the lyse is quenched and the bound RBC's are magnetically removedand then the WBC's are analyzed as previously described.

Referring now to FIGS. 17A and 17B, two sets of results depicted inscattergrams obtained from a whole blood sample utilizing a prototypeanalyzer similar to the instrument 360 are illustrated. Two WBCpopulations are removed and the T₈ subset is directly analyzed. The T₈subset is the cells or formed bodies which have the receptor or antigento which the T₈ specific antibody binds to. In the Figures., these aredesignated as T₈ ⁺. The cells or formed bodies which do not have thereceptor or antigen to which the T₈ specific antibody binds to aredesignated as T₈ ⁻. In these examples, the biological medium 342 was a20 microliter sample of whole blood utilized with the mixer 376. In bothFIGS. 17A and 17B, the 20 microliter sample of whole blood, medium 342,was combined with 40 microliters of magnetic microspheres with the RBCspecific antibody bound thereto, combined with 120 microliters of buffersolution and 10 microliters of magnetic microspheres with an N and Especific antibody bound thereto, combined with 30 microliters of buffersolution which together form the reactant 346. One such exemplary N andE specific antibody is disclosed in U.S. Ser. No. 068,618, entitledMONOCLONAL ANTIBODY SPECIFIC TO A COMMON DETERMINANT SITE OF NEUTROPHILSAND EOSINOPHILS, filed Jun. 3, 1987, now U.S. Pat. No. 4,865,971 whichis incorporated herein by reference.

The magnetic microspheres can be of any suitable type and in the exampleare polystyrene magnetic microspheres of 0.7 micron diameter, with aweight to volume of 10% solids, sold by Seradyn, Inc. of Indianapolis,Ind. The reaction mixture then was mixed in the mixer 376 for 10seconds, placed in the magnetic field of the magnet 440 for 15 secondsand then the resulting mixture with the RBC's, E's and N's removed wasanalyzed in the analyzer 396. The resulting scattergram A is illustratedin FIG. 17A.

The scattergram of FIG. 17B results from the same procedure with theaddition of 12.5 microliters of non-magnetic microspheres with a T₈specific antibody bound thereto combined with 12.5 microliters of buffersolution to form the reactant 346. The T₈ specific antibody is soldunder the Trademark COULTER CLONE@ by Coulter Immunology Division ofCoulter Corporation. The non-magnetic microspheres again can be of anysuitable type and in the examples are surfactant free sulfatedpolystyrene latex microspheres of 1.78 micron diameter with a weight tovolume of 8% solids, sold as IDC microspheres by Interfacial Dynamics ofPortland, Oreg.

The addition of the T₈ microspheres shifts the bound CD8 cells to anarea B where they separately can be identified and counted as seen bycomparing the scattergram of FIGS. 17A and 17B. In FIG. 17A the CD8cells are hidden by the remaining WBC's. The N's and E's are removedfrom the scattergrams or they would obscure the identification of theshifted CD8 cells in FIG. 17B. FIG. 17A illustrates the removal of theN's and E's, while FIG. 17B then clearly illustrates the shift of theCD8 bound cells from area A to area B. The buffer solution can bephosphate buffered saline sold by Sigma Chemical Company of St. Louis,Mo.

FIG. 18A further illustrates the normal scattergram or 3 parameterhistogram positioning of the M, L and G cell populations from theanalyzer 352. Without removal of the G's, as seen in FIG. 17B, the areaB of the shifted WBC subset would be obscured by the G's, which are farmore numerous in number. FIG. 18B is a scattergram illustrating the WBCpopulations M, L and B remaining after removal of the E's and N's.Although the B's still may partially obscure the area of interest, theirpercentage number of the WBC populations is of a small enough order tonot substantially affect the desired calculation of the subset,percentage. However, the B's contribution can be subtracted from thesubset percentage if so desired.

Referring now to FIGS. 19A-D, 20A-D and 21A-D, the direct subsetanalysis of the CD2, CD4, CD8 and CD20 WBC subset populations ofrespective samples from three different patients is illustrated. In thecase of each subset population, 28 microliters of a whole blood samplewas combined with 20 microliters of magnetic microspheres (2.5% weightper volume solution) with the N and E specific antibody bound thereto.In addition, non-magnetic microspheres with the respective monoclonalantibody for the respective WBC subset are also combined with thesample. The respective amounts of T₄, T₈, T₁₁ or B₁ coated microspheresare 40 microliters each. (1% weight per volume solution for each one).Each respective total mixture, i.e., N and E microspheres with T₈, forexample, is combined with a buffer solution of phosphate bufferedsaline, 1% bovine serum albumin, pH of 7.2 to 7.4 for a total volume of150 microliters. Each respective mixture is mixed in the chamber 378 bythe mixer 376 for two minutes and then placed in the magnetic field 440for one minute. In these examples, the RBC's are removed sequentiallyutilizing the lyse above referred to. The WBC microspheres are firstadded, then the RBC's are removed by lysing with 300 microliters oflyse, such as Erythrolyse lytic reagent sold by Coulter Electronics,such as from the lyse source 370. The mixture then is quenched with 120microliters of quench, such as Stabilyse, a leukocyte preservative alsosold by Coulter Electronics, from the source 380 and then fed to theanalyzer 396 for analysis.

The right-hand block (1) in each scattergram represents the respectiveWBC subset population of interest. The blocks 1, 2, 3, etc. illustratedin the Figures are visually or automatically fit around the WBCpopulation or subset of interest.

The results were compared utilizing conventional flow cytometry and gavethe following comparative results in percentages for the three samplesby the method of the invention (SHIFT) vs. flow cytometry (CYT).

    ______________________________________                                        T.sub.4       T.sub.8   T.sub.11  B.sub.1                                     (FIG. 19A)    (FIG. 19B)                                                                              (FIG. 19C)                                                                              (FIG. 19D)                                          Shift  CYT    Shift                                                                              CYT  Shift                                                                              CYT  Shift                                                                              CYT                            ______________________________________                                        Patient 51     52     18   22   82   76   15   13                             Sample 1                                                                      ______________________________________                                        T.sub.4       T.sub.8   T.sub.11  B.sub.1                                     (FIG. 20A)    (FIG. 20B)                                                                              (FIG. 20C)                                                                              (FIG. 20D)                                          Shift  CYT    Shift                                                                              CYT  Shift                                                                              CYT  Shift                                                                              CYT                            ______________________________________                                        Patient 53     54     32   29   89   83   6.5  7.5                            Sample 2                                                                      ______________________________________                                        T.sub.4       T.sub.8   T.sub.11  B.sub.1                                     (FIG. 21A)    (FIG. 21B)                                                                              (FIG. 21C)                                                                              (FIG. 21D)                                          Shift  CYT    Shift                                                                              CYT  Shift                                                                              CYT  Shift                                                                              CYT                            ______________________________________                                        Patient 46     46     24   18   86   81   11   10                             Sample 3                                                                      ______________________________________                                    

FIG. 22A also illustrates the normal scattergram or 3 parameterpositioning of the M, L and G cell populations from the analyzer 352.Without removal of the N's and E's, the CD4 cell population would beobscured. By shifting the N's and E's with the N and E specificmonoclonal antibody microspheres to an area or block 1 illustrated inFIG. 22B, the CD4 population can be shifted and viewed in the block orarea 2. This area would have been obscured by the N's and E's as seen inFIG. 22A. In this example for FIG. 22C, 28 microliters of a whole bloodsample were combined with 50 microliters of 2.2 micron microspheres withthe N and E specific monoclonal antibody bound thereto and 50microliters of microspheres with T₄ specific monoclonal antibody boundthereto and 22 microliters of diluent. FIG. 22B was the same without theT₄ microspheres and with 72 microliters of diluent and FIG. 22A was thesame without any microspheres and 122 microliters of diluent. Referringto FIGS. 23A-D, direct WBC analysis utilizing a plurality ofmicrospheres bound to the WBC subset of interest is illustrated. FIGS.23A and 23B respectively illustrate scattergrams of only the Lpopulation with the T₄ WBC subset and the T₁₁ WBC subset each shiftedwith 0.8 micron non-magnetic microspheres. The shift is insufficient todifferentiate the WBC subset population in FIGS. 23A and 23B. FIGS. 23Cand 23D respectively illustrate scattergrams of only the L populationwith the T₄ WBC subset and the T₁₁ WBC subset shifted by being bound toboth a 0.8 micron and a 2.2 micron microsphere. The 2.2 micronmicrosphere is bound to the 0.8 micron microsphere by having Goatanti-mouse IgG antibody bound thereto, which binds to the T₄ or T₁₁antibody bound to the 0.8 micron microsphere.

The effect of the size of the non-magnetic microsphere bound to the WBCsubset of interest is illustrated in FIGS. 24A-C. In this example, a 28microliter sample of whole blood was combined with 10 microliters ofmagnetic microspheres having the N and E specific antibody bound thereto(2.5% weight per volume solution) and 40 microliters of non-magneticmicrospheres having the T₈ specific antibody bound thereto (1% weightper volume solution). The T₈ microspheres were of two different sizes toillustrate the difference in the shift on the scattergram. A buffersolution again was added to form a mixture volume of 150 microliters.The mixture was mixed for 2 minutes and placed in the magnetic field for1 minute. The resultant N and E removed mixture then was lysed to removethe RBC and then analyzed. FIG. 24A illustrates a control WBC subsetwithout a microsphere attached thereto, a T₈ WBC subset with a 2.2micron non-magnetic microsphere bound thereto and a T₈ WBC subset with a3.0 micron non-magnetic microsphere bound thereto. The width and heightillustrate the standard deviation of the detected signal. FIG. 24B is ascattergram illustrating the T₈ WBC subset shift with the 3.0 micronmicrospheres bound thereto, while FIG. 24C is a scattergram illustratingthe T₈ WBC subset shift with the 2.2 micron microspheres bound thereto.The analyzed percentage of the T₈ WBC subset for the differentmicrospheres were respectively, 20.9 and 19.3. The larger microsphereclearly generated a more distinct scattergram pattern as illustrated byFIG. 24B.

Referring now to FIGS. 25A-D, the simultaneous direct analysis of twoWBC subset populations is illustrated in accordance with the secondparent application. In this example, 28 microliters of a whole bloodsample was combined with 10 microliters of magnetic microspheres havingthe N and E specific antibody bound thereto, 52 microliters of buffersolution and 40 microliters of non-magnetic 3.0 micron microspheres withthe T₈ specific antibody bound thereto and mixed for 2 minutes. Themixture then was placed in the magnetic field for 1 minute and then theresultant N and E removed mixture was lysed to remove the RBC's and thenanalyzed. FIG. 25A illustrates a control WBC subset sample without amicrosphere bound thereto, a T₄ reading with a 2.2 micron non-magneticmicrosphere bound thereto and a T₈ reading with a 3.0 micronnon-magnetic microsphere bound thereto. This illustrates the separationbetween the two shifted WBC subset populations. FIG. 25B is ascattergram analysis with only the T₄ WBC subset population bound to the2.2 micron microspheres shifted to area A and FIG. 25C is a scattergramanalysis with only the T₈ WBC subset population bound to the 3.0 micronmicrospheres shifted to area B. FIG. 25D illustrates a scattergramanalysis with both the T₄ and T₈ WBC subset populations shifted to therespective areas A and B. This allows a simultaneous analysis of boththe T₄ and T₈ subset populations.

Referring now to FIGS. 26A-D, three populations of L's, M's and G's areillustrated on four different scattergrams utilizing differentparameters. Although the previous examples have been illustratedutilizing DC vs. opacity (RF/DC), the scattergrams can be formedutilizing virtually any two different parameters. FIG. 26A illustrates ascattergram utilizing DC vs. RF alone, FIG. 26B utilizes RF vs. opacity,FIG. 26C utilizes DC-RF vs. opacity and FIG. 26D utilizes DC vs. opacityas previously illustrated. Further, although DC vs. RF or RF/DC has beenutilized, any two different frequencies are adequate as long as thesignals are separable from each other, because of their frequencyspectrum location and/or the difference in phase relationship. Opacityis a preferable parameter since it essentially is a normalization of theRF signal. Clearly, as illustrated in FIGS. 26A-D, the presentation ofthe data can be varied as desired. DC is a function of volume of thecell or formed body sensed, while RF is a function of the internalconductivity and volume of the sensed cell or formed body.

Referring now to FIGS. 27-46, the embodiments of the third parentapplication, Ser. No. 339,156 are illustrated.

Referring to FIG. 27, a first embodiment of a method and apparatus forperforming classification of cells such as a multipart differential isdesignated generally by the reference numeral 500. The instrument oranalyzer 500 includes a biological sample 502, which contains at least afirst set of viable biological cells (not illustrated), including atleast two white blood cell populations, such as in or from a whole bloodsample.

The cells of the biological sample 502 are to be involved in abiological reaction in a quantitative and/or qualitative determinationor analysis. The biological sample 502 can include a buffer into whichthe cells are added.

The biological sample 502 is combined via a line 504 with at least onereactant 506 via a line 508. In the analyzer 500, the RBC's are removedfrom the mixture at an RBC removing station 510. As stated in the firstparent application, the RBC's can be removed from the station 510 in anumber of ways, such as enumerated with respect to the station 20.

A first portion of the mixture with the RBC's removed, then is fed to aWBC analyzer 512 via a line 514. This obtains a standard or control forthe total or whole WBC populations of the biological sample 502. Theanalyzer 512 can be the same as the analyzer 86 or can be a lightsensing analyzer, such as described in U.S. Ser. No. 025,442 filed Mar.13, 1987 now abandoned and U.S. Ser. No. 129,954, filed Dec. 4, 1987,now abandoned in favor of continuation application U.S. Ser. No.479,199, filed Feb. 13, 1990 now U.S. Pat. No. 5,125,737 entitledMULTI-PART DIFFERENTIAL ANALYZING APPARATUS UTILIZING LIGHT SCATTERTECHNIQUES, which are incorporated herein by reference. The singlesensing parameter can be electronic, such as RF or DC or light, such asmedian angle light scatter (Scatter) or any other desired lightparameter.

A second portion of the mixture is fed to a N removing station 516 via aline 518. The N's are removed by the addition of the appropriatemagnetic microspheres with the N specific antibody bound thereto. Inthis example, the particular N specific antibody utilized is disclosedin Case 168,306, MONOCLONAL ANTIBODY SPECIFIC TO NEUTROPHILS, filed Dec.8, 1986, now U.S. Ser. No. 938,864, which is incorporated herein byreference. A magnet or magnetic field is utilized, as before discussed,to remove the magnetically bound cells from the mixture. The remainingmixture with the N's removed then is fed via a line 520 to the analyzer512. The analyzed results of this portion of the mixture then can becompared with the analyzed results of the first mixture portion in acomparator 522 to obtain the percentage of N's in the biological sample502.

A third portion of the mixture is fed to a N and E removing station 524via a line 526. The N's and E's are removed by the addition ofappropriate magnetic microspheres with the N and E specific antibodybound thereto. One such exemplary N and E specific antibody is disclosedin U.S. Ser. No. 068,618, entitled MONOCLONAL ANTIBODY SPECIFIC TO ACOMMON DETERMINANT SITE OF NEUTROPHILS AND EOSINOPHILS, filed Jun. 3,1987, which is incorporated herein by reference. Separate N and Especific antibodies, bound to the same or separate magneticmicrospheres, also can be utilized, where appropriate or as developed.The remaining mixture with the N's and E's removed then is fed via aline 528 to the analyzer 512. The analyzed results of this portion ofthe mixture then is compared to the results of the first and secondmixture portions in the comparator 522 to obtain the percentage of E'sand M's in the biological sample 502.

A fourth portion of the mixture is fed to a L and N removing station 530via a line 532. The L's and N's are removed by the addition of theappropriate magnetic microspheres with the L and N specific antibodybound thereto. The N specific antibody can be either of the abovereferenced N specific antibodies, or other appropriate antibodies. The Lspecific antibody can be an L specific antibody as developed or acombination of the specific antibodies sold under the nomenclature T11and 2H4 by Coulter Immunology Division of Coulter Corporation, whichtogether bind all the L's. The remaining mixture with the L's and N'sremoved then is fed via a line 534 to the analyzer 512. The analyzedresults of this portion of the mixture then can be compared to theresults of the other mixture portions to obtain the percentage of B'sand L's in the biological sample 502.

Thus, the analyzer 500 performs a single classification of cells, suchas N's utilizing lines or channels 514 and 518, E's and/or N's and/orM's utilizing lines 514, 518 and 526 and a full five part WBCdifferential utilizing all four lines. One most important feature of theanalyzer 500 is that the mixtures can be analyzed utilizing only asingle analyzing parameter, such as one electronic parameter or onelight parameter. Other combinations can be utilized, but in each caseonly a single sensed parameter or characteristic is necessary to performthe classification of the parent application.

FIG. 28 illustrates a specific analyzing instrument embodimentincorporating the method and apparatus of the analyzer 500 designatedgenerally by the reference numeral 540. The instrument 540 includes anaspirator pumping mechanism 542 which is utilized to draw the biologicalsample of interest, for example the sample 502, into the instrument 540.The aspirator 542 is coupled via a line 544 to a sampling valve 546,which can be coupled to a sample probe 548. The biological sample 502then is fed via a line 550 and a multipart valve 551 into the separatechannels 514, 518, 526 and 532.

Describing first the channel 514, the sample portion is fed to a chamber552. The chamber can be a mixing chamber into which the sample andreactant is fed to remove the RBC's. For example, utilizing lyse, theRBC's are lysed in the chamber 552 by adding the appropriate lysethereto and preferably mixing therewith, then when the reaction iscompleted, a quench or fix is supplied to the chamber 552.

Specific details of an appropriate mixing apparatus, which can beutilized herein are disclosed in Ser. No. 025,337, filed Mar. 13, 1987,entitled METHOD AND APPARATUS FOR RAPID MIXING OF SMALL VOLUMES FORENHANCING BIOLOGICAL REACTIONS, which is incorporated herein byreference. By utilizing the mixer, the reactions are greatly enhanced inspeed without significantly damaging the properties of interest of thecells, such as, can occur by raising the reaction temperature. Further,the reactions generally are completed in significantly less than aminute, generally on the order of fifteen seconds or less. This allows arapid analysis of the automatic high volume analyzer instrument 540.

The quenched reactant mixture with the RBC's removed by the lyse then isfed via a line 554 to a multipart valve 556, or directly to a WBCanalyzer 558 via a line 560. The analyzer 558 can be of many physicaltypes in accordance with the counting and sizing techniques described byWallace H. Coulter in U.S. Pat. No. 2,656,508 utilizing light orelectronic sensing as embodied in the numerous commercial blood cellcounters of the assignee, Coulter Electronics, Inc.

The analyzer 558, in general, includes a flow sensor or sensing chamber562. The chamber 562 includes a transducer 564 which has an aperturetherethrough. The chamber 562 can include a first portion 566 which hasa first electrode 568 in contact with the fluid therein. The chamberportion 566 and the electrode 568 can communicate through the sensingaperture with a second chamber portion 570 having a second electrode 572therein.

The electrodes 568 and 572 are coupled via reactive leads to an RF/DCsource and sensing circuit 574. The circuit 574 couples either or both aDC, or low frequency current or signal, and a high frequency signalbetween the electrodes 568 and 572.

The low frequency signal is utilized to sense the amplitude of a signalpulse caused by a cell passing through the sensing aperture. The highfrequency signal can be utilized in the same manner as a singleparameter or with the low frequency signal to obtain the electricalopacity of the same cell passing through the sensing aperture.

The measuring of the electrical opacity of cells was described byWallace H. Coulter and Walter R. Hogg in U.S. Pat. No. 3,502,974 andseveral patents and publications of the assignee, Coulter Electronics,Inc., since that patent. One specific circuit which can be utilizedherein is disclosed in Case 168,008, entitled PARTICLE ANALYZER FORMEASURING THE RESISTANCE AND REACTANCE OF A PARTICLE, filed Oct. 21,1986 as U.S. Ser. No. 921,654, now U.S. Pat. No. 4,791,355, which isincorporated herein by reference.

The signals generated by the circuit 574 from the sensed cells arecoupled via a DC signal lead 576 and/or an RF signal lead 578 to acomparator 580 (like the comparator 26). The comparator 580 can hold thesignal generated from the first portion, i.e., those without the WBCpopulation or population subset subtracted, for a comparison with theresults from the subsequent portions described hereinafter. The analyzer558 can include a sheath flow to focus the cells in the sensor 562, inthe well known manner. The sheath flow can be provided by a fluidicsystem 582, coupled to the sensor in a known manner.

The analyzer 558 has been illustrated with both RF and DC analyzingcircuitry for example purposes only. In obtaining the multipart WBCpopulation or subset population characterization of the parentapplication, only a single sensing parameter, electronic or optical,need be utilized. In the case of electronic sensing, the parameter canbe either DC or RF. Utilizing optical sensing, not illustrated, againonly a single parameter such as median angle light scatter need beutilized. This one dimension sensing can simplify the instrument 540, aswell as decrease the cost thereof.

A second portion of the sample mixture is fed through the valve 551 viathe line 518 to the N removing station 516. The station 516 includes amixer or chamber 584. The mixing chamber 584 has the second mixtureportion fed thereto via the line 518. The mixer 584 includes all of thevarious options above described and, for example, includes a lyse inputline for the RBC lyse.

When the lyse is utilized, after mixing as illustrated functionally at586, then the quench is added via a quench line. At the same time orsequentially, the N's are being removed by the addition of theappropriate magnetic microspheres with the N specific antibody boundthereto from a source of microspheres 588 fed to the chamber 584 via aline 590. A magnet 592 or magnetic field is then utilized to remove themagnetically bound cells on the magnetic microspheres. The mixed andquenched mixture than is fed via the line 520 through the valve 556 andthe line 560 to the WBC analyzer 558 to be analyzed as before described.The analyzed mixture with the N's removed then is compared in thecomparator 580 to determine the percentage of N's in the sample 502.

A third mixture portion of the sample 502 is fed via the line 550 andthe valve 551 via the line 526 to the N and E removing station 524. Thestation 524 includes a mixing chamber 594. In the third portion, theRBC's are removed by the RBC lyse fed into the chamber 594. The lyse ismixed with the sample portion and then a quench is added via a quenchline. The N's and E's are removed by magnetic microspheres having the Nand E specific antibody or antibodies bound thereto from a microspheresource 596 fed into the chamber 594 via a line 598. The microspheres aremixed, functionally at 600, and then the bound N and E microspheres aremagnetically removed, functionally at 602. The N and E removed mixturethen is fed via the line 528 to the valve 556 and via the line 560 tothe analyzer 558 to also obtain the above mentioned results.

The instrument can be utilized with the first two channels 514 and 518to determine the N percentage or with the first three channels 514, 518and 526 to obtain the N's and E's percentages and/or the M's percentage,as desired. To obtain further WBC population or subset populationcharacterizations, a fourth portion of the sample mixture 502 is fed viathe line 550 and the valve 551 via the line 532 to the L and N removingstation 530. Again, the RBC's are removed such as by lysing in a chamber604, and the L's and N's are removed by binding the L's and N's tomagnetic microspheres having the L and N specific antibody or antibodiesbound thereto from a source 606 fed into the mixing chamber 604 via aline 608. The microspheres are mixed, functionally at 610, and then themagnetically bound L and N microspheres are magnetically removed,functionally at 612.

The L and N removed mixture then is fed via the line 534 to the valve556 and via the line 560 to the analyzer 558 to obtain theabove-mentioned results. Utilizing combinations of the results from theother channels, the percentage of L's and/or B's then can be obtainedsuch as to perform a full five part WBC differential to obtain thepercentage of N's, E's, M's, L's and B's. The mixers include appropriaterinse lines and waste lines and the instrument 540 can include a proberinse 614 to cleanse the instrument 540 prior to aspirating the nextsample or sample portion for analyzing. Further, the sample 502 can bediluted from a source 616 via a line 618 if desired.

Only one specific hardware embodiment incorporating the method andapparatus of the analyzer 500 has been illustrated, but like theembodiments in the parent application, the analyzing instrument 540 canbe implemented in numerous configurations. For example, the analyzer 540could include a single channel, such as the channel 518 and the portionseach can be run sequentially through the station 516 with theappropriate WBC population or populations removed from each portion, aspreviously described with respect to the separate channels.

Referring now to FIGS. 29A-D and FIGS. 30A-D, two sets of onedimensional scattergram multipart characterization results areillustrated, obtained from a whole blood sample, utilizing a prototypeanalyzing method similar to the analyzer instrument 540. The biologicalsample in each case was a 28 microliter sample of whole blood, which wascombined with 122 microliters of buffer solution for the sample portionutilized in the first channel 514. The sample portion was lysed with 300microliters of the RBC preferential lyse above referenced in the chamber552. The sample portion was lysed for 4 seconds and then quenched with120 microliters of quench before being fed to the analyzer 558 via theline 554, the valve 556 and the line 560. The results of analyzingutilizing a one dimensional electronic sensing parameter are illustratedin FIGS. 29 and 30. DC was utilized to obtain the data in FIGS. 29A-29Dand RF was utilized to obtain the data (for comparison) in FIGS.30A-30D, utilizing the same sample portion and measured at the same timein the analyzer 558. This results in two clearly identifiable data peaks620 and 622 in FIG. 29A and peaks 624 and 626 in FIG. 30A. Peaks 620 and624 are indicative of the percentage of L's and B's in the sample. Peaks622 and 626 are indicative of the percentage of N's, E's and M's.Clearly, in one dimension, without further manipulation, the individualpercentages are masked by the competing cells in the same data peak. Asdescribed in the above referenced parent application, this is not aproblem, at least for some cells when greater than one parameter isutilized to differentiate the data.

In the second channel 518, a second whole blood sample portion of 28microliters is combined with 40 microliters of magnetic microsphereswith the N specific antibody bound thereto and combined with 82microliters of buffer solution in the chamber 584. The sample portionwas mixed for 60 seconds, lysed and quenched in the same manner as inthe channel 514 and then placed in the magnetic field 592 for 30 secondsbefore the mixture with the N's removed is fed to the analyzer 558 viathe line 520, the valve 556 and the line 560. This results in two datapeaks 628 and 630 in FIG. 29B and two data peaks 632 and 634 in FIG.30B. Peaks 628 and 632 remain the same as the peaks 620 and 624 althoughtheir percentage of the sample mixture portion now is greater, while thepeaks 630 and 634 are indicative of the percentage of the E's and M'swith the N's removed. The data peaks 630 and 634 can then be comparedwith the respective data peaks 622 and 626 to determine the percentageof N's in the whole blood sample.

Next, or in any order including substantially simultaneously, a third 28microliter sample portion is fed to the third channel 526 and mixed with20 microliters of magnetic microspheres with the E and N specificantibody or antibodies bound thereto and combined with 102 microlitersof buffer solution in the chamber 594. The sample portion was mixed for30 seconds, lysed and quenched in the same manner as in the channel 514and then placed in the magnetic field 602 for 30 seconds before themixture with the N's and E's removed is fed to the analyzer 558 via theline 528, the valve 556 and the line 560. This results again in two datapeaks 636 and 638 in FIG. 29C and data peaks 640 and 642 in FIG. 30C.Peaks 636 and 640 again are the same contribution as the peaks 620 and624, while the data peaks 638 and 642 are indicative of the percentageof the M's in the whole blood sample as compared to the data peaks 622and 626. The data peaks 638 and 642 then can be compared to the datapeaks 630 and 634 also to determine the percentage of E's in the wholeblood sample.

A fourth 28 microliter sample portion is fed to the fourth channel 532and mixed with 70 microliters of magnetic microspheres with a CD2specific antibody bound thereto and 50 microliters of magneticmicrospheres with a CD45R specific antibody bound thereto in the chamber604. The sample portion was mixed for 2 minutes and then placed in themagnetic field 612 for 30 seconds and then the remaining mixture isremoved to a holding chamber 644 via a line 646. The holding chamber 644currently appears to be a necessary operation, because the specificantibodies utilized as above referenced under the nomenclature T11 and2H4 and the N specific antibody, appear to interfere with one anotherwhen utilized simultaneously. It is not known whether this is due to thespecific antibodies or to the nature of the cells themselves. Once themixture with the T11 and 2H4 bound cells removed is fed into the holdingchamber 644, the chamber 604 then is rinsed with the magnetic fieldremoved to remove the magnetic microspheres having the CD2 and CD45Rcell clusters bound thereto.

The N bound magnetic microspheres then are isolated in the chamber 604,which can be provided by another source, other than the source 606 (notillustrated). The isolation is accomplished by holding the magneticmicrospheres with 40 microliters of the N specific antibody boundthereto in the magnetic field 612 and removing the buffer solution towaste. Alternately, the mixture can be adjusted so that the buffersolution is utilized as part of the mixture or a concentrated volume ofmagnetic microspheres can be utilized and the solution need not beutilized. The sample portion then is returned to the chamber 604 fromthe chamber 644 via a line 648 and mixed with the magnetic microsphereswith the N specific antibody bound thereto, lysed and quenched as in thechannel 514 and then again placed in the magnetic field 612 for 30seconds before the mixture with all the L's and N's removed is fed tothe analyzer 558 via the line 534, the valve 556 and the line 560. Thisresults in two data peaks 650 and 652 in FIG. 29D and two data peaks 654and 656 in FIG. 30D. Peaks 650 and 654 represent only the B's, since allthe L's have been removed. Since the L's are about 30 percent of anormal whole blood sample, this enhances the B's which originally areabout 1 percent of the sample to a very significant data peak (i.e.,amount of data). The B's are further enhanced since the N's also wereremoved from the peaks 652 and 656 and they originally are about 60percent of the sample. Hence, the B's now represent about 10 percent ofthe remaining B, M and E cells.

The actual percentage calculations are performed as follows, with thedata peak numbers calculated by dividing the data peak (number of cellscounted in the peak) by the total data from both peaks (utilizing FIGS.29A-29D for example purposes):

1) M %-determined from channel 526:

    [(Pk1 (620)) (Pk1 (636))] xx Pk2 (638)=M %

2) M+E %-determined from channel 518:

    [(Pk1 (620)) (Pk1 (628))] xx Pk2 (630)=M+E %

3) E %=M+E %-M %

4) N %=(Pk2 (622))-M+E %

5) B %-determined from channel 532:

    [(Pk2 (630)) (Pk2 652))] xx (Pk1 (650))=B.sup.1 %

    B.sup.1 % xx [(P1 (620)) (Pk1 (628))] =B %

6) L %=(Pk1 (620))-B %

For the data illustrated in FIGS. 29 and 30, the calculations from theRF and DC data are set forth in Table II.

                  TABLE II                                                        ______________________________________                                                 DC               RF                                                  Channel    Pk1    Pk2         Pk1  Pk2                                        ______________________________________                                        514        28.1   71.9        30.3 69.7                                       518        74.4   25.6        74.1 25.9                                       526        83.8   16.2        83.4 16.6                                       532        10.9   90.1        10.0 90.0                                       ______________________________________                                    

A full five part differential in percentage of the N's, L's, M's, E'sand B's also was calculated from the DC and RF data peaks and comparedto a light sensing instrument for verification purposes, such asdescribed in U.S. Ser. No. 025,442.

                  TABLE III                                                       ______________________________________                                        5 PART DIFFERENTIAL                                                           LIGHT          DC      RF                                                     ______________________________________                                        N 61.58        N 62.2  N 59.1                                                 L 28.50        L 26.9  L 29.1                                                 M  6.27        M  5.4  M  6.0                                                 E  3.10        E  4.3  E  4.6                                                 B  0.56        B  1.2  B  1.2                                                 ______________________________________                                    

Referring now to FIGS. 31A-D and FIGS. 32A-D, a second two sets of onedimensional scattergram multipart characterization results areillustrated, obtained from a second whole blood sample, again utilizinga prototype analyzing method similar to the analyzer instrument 540. Thebiological sample in each case again was a 28 microliter sample of wholeblood, prepared for each channel as described with respect to FIGS. 29and 31. The sample portion was lysed in the first channel 514 in thechamber 552 and then quenched before being fed to the analyzer 558. Theresults of analyzing utilizing a one dimensional electronic sensingparameter are illustrated in FIGS. 31 and 32. DC was utilized to obtainthe data in FIGS. 31A-31D and RF was utilized to obtain the data (forcomparison) in FIGS. 32A-32D, utilizing the same sample portion andmeasuring at the same time in the analyzer 558. This results in twoclearly identifiable data peaks 658 and 660 in FIG. 31A and peaks 662and 664 in FIG. 32A. Peaks 658 and 662 are indicative of the percentageof L's and B's in the sample. Peaks 660 and 664 are indicative of thepercentage of N's, E's and M's. Again, in one dimension, without furthermanipulation, the individual percentages are masked by the competingcells in the same data peak.

In the second channel 518, a second whole blood sample portion iscombined with the magnetic microspheres with the N specific antibodybound thereto in the chamber 584, mixed, lysed and quenched and thenplaced in the magnetic field 592 before the mixture with the N's removedis fed to the analyzer 558. This results in two data peaks 666 and 668in FIG. 31B and two data peaks 670 and 672 in FIG. 32B. Peaks 666 and670 remain the same as the peaks 568 and 662 although their percentageof the sample mixture portion now is greater, while the peaks 668 and672 are indicative of the percentage of the E's and M's with the N'sremoved. The data peaks 668 and 672 can then be compared with therespective data peaks 660 and 664 to determine the percentage of N's inthe whole blood sample.

Next, or in any order including substantially simultaneously, a thirdsample portion is fed to the third channel 526 and mixed with themagnetic microspheres with the E and N specific antibody or antibodiesbound thereto in the chamber 594, mixed, lysed and quenched and thenplaced in the magnetic field 602 before the mixture with the N's and E'sremoved is fed to the analyzer 558. This results again in two data peaks674 and 676 in FIG. 31C and data peaks 678 and 680 in FIG. 32C. Peaks674 and 678 again are the same contribution as the peaks 658 and 662,while the data peaks 676 and 680 are indicative of the percentage of theM's in the whole blood sample as compared to the data peaks 660 and 664.The data peaks 676 and 680 then can be compared to the data peaks 668and 672 also to determine the percentage of E's in the whole bloodsample.

A fourth sample portion is fed to the fourth channel 532 and mixed withthe magnetic microspheres with a CD2 specific antibody bound thereto andthe magnetic microspheres with a CD45R specific antibody bound theretoin the chamber 604, mixed, then placed in the magnetic field 612 andthen the remaining mixture is removed to the holding chamber 644.

The N bound magnetic microspheres then are isolated in the chamber 604by holding the magnetic microspheres with the N specific antibody boundthereto in the magnetic field 612 and removing the buffer solution. Thesample portion then is returned to the chamber 604 after it is rinsedfrom the chamber 644 via a line 648 and mixed with the magneticmicrospheres with the N specific antibody bound thereto, lysed andquenched and then again placed in the magnetic field 612 before themixture with all the L's and N's removed is fed to the analyzer 558.This results in two data peaks 682 and 684 in FIG. 31D and two datapeaks 686 and 688 in FIG. 32D. Peaks 682 and 686 represent only the B's,since all the L's have been removed to enhance the B's to a verysignificant data peak (i.e., amount of data). The B's are furtherenhanced since the N's also were removed from the peaks 684 and 688 andhence from the remaining sample portion.

The actual percentage calculations are performed as before as set forthhereinafter, with the data peak numbers calculated by dividing the datapeak (number of cells counted in the peak) by the total data from bothpeaks (utilizing FIGS. 31A-31D for example purposes):

1) M %-determined from channel 526:

    [(Pk1 (658)) (Pk1 (674))] xx Pk2 (676)=M %

2) M+E %-determined from channel 518:

    [(Pk1 (658)) (Pk1 (666))] xx Pk2 (66)=M+E %

3) E %=M+E %-M %

4) N %=(Pk2 (660))-M+E %

5) B %-determined from channel 532:

    [(Pk2 (668)) (Pk2 (684))] xx (Pk1 (682)=B.sup.1 %

    B.sup.1 % xx [(Pk1 (658)) (Pk1 (666))] =B %

6) L %=(Pk1 (658))-B %

For the data illustrated in FIGS. 31 and 32, the calculations from theRF and DC data are set forth in Table IV.

                  TABLE IV                                                        ______________________________________                                                 DC               RF                                                  Channel    Pk1    Pk2         Pk1  Pk2                                        ______________________________________                                        514        24.0   76.0        26.0 74.0                                       518        63.3   36.7        62.6 37.4                                       526        76.0   24.0        75.2 24.8                                       532        10.6   89.4         9.7 90.3                                       ______________________________________                                    

A full five part differential in percentage of the N's, L's, M's, E'sand B's again was calculated from the DC and RF data peaks and againcompared to a light sensing instrument for verification purposes, suchas described in U.S. Ser. No. 025,442.

                  TABLE V                                                         ______________________________________                                        5 PART DIFFERENTIAL                                                           LIGHT          DC      RF                                                     ______________________________________                                        N 60.37        N 62.1  N 58.5                                                 L 24.70        L 22.4  L 24.3                                                 M  8.62        M  7.6  M  8.6                                                 E  5.12        E  6.3  E  6.9                                                 B  1.18        B  1.6  B  1.7                                                 ______________________________________                                    

The calculations are set forth below utilizing the DC data peaks:##EQU1##

The data peaks in FIGS. 29-32 were depicted utilizing a singleelectronic sensing parameter, either DC or RF. Opacity was notillustrated, but also could be utilized if desired, since it is RF/DC aspreviously described. A single light sensing parameter also can beutilized, for example, median angle light scatter (Scatter), asillustrated in FIGS. 33-36.

Referring to FIG. 33, the M's, L's, N's and E's groups of cells areclearly separated by utilizing two parameters, Scatter and electricalvolume. The B's are obscured in this data pattern. However, in onedimension, the same scatter data illustrated in FIG. 34 now results inthe M's obscuring the L's or vice versa as seen in data peak 690. TheN's and E's are illustrated in data peak 692 and the E's in data peak693, representing the same data as in FIG. 33.

One method of solving the problem of the obscured data in the data peak690 is to remove the M's. Currently this would be accomplished in anoffline or prepreparation mode, since the M's are removed slowly whenutilizing a CD14 specific antibody, such as M02 sold by CoulterImmunology Division of Coulter Corporation. For example, 400 microlitersof a whole blood sample are combined with 200 microliters of a 21/2percent solution of magnetic microspheres with the CD14 specificantibody bound thereto. The magnetic microspheres are first isolated byremoving the fluid therefrom while holding the microspheres in amagnetic field. The sample then is added and the mixture is gentlymixed, such as by rocking for about 30 minutes and then placed in amagnetic field for about 5 minutes after which the preprepared mixture,with the M's removed is analyzed in the instrument 540 as previouslydescribed. The data is illustrated again in two dimensions forcomparison purposes in FIG. 35, where it clearly can be seen that theM's have been depleted by comparison with FIG. 33, enhancing theremaining WBC population data.

The one dimensional scatter data is illustrated in FIG. 36, againresulting in two data peaks 694 and 696. The peak 694 is the data peak690 with the M's removed, leaving only the L's. The peak 696 is the sameas the data peak 692, and peak 697 is the same as peak 693, both peaks696 and 697 are enhanced by the removal of the M's from the sampleportion. Although not specifically illustrated, the sample mixture withthe M's removed as illustrated in peak 694, now can be further depletedto perform L subset analyses, as will be further described hereinafter.

Although the analyzer 500 is illustrated for explanation purposesutilizing four separate channels, this only enhances the speed of theclassification method of the parent application. The parent applicationalso can be practiced utilizing a single channel, with the differentportions of the biological sample 502 being processed sequentially.

FIG. 37 illustrates an analyzer embodiment for a method and apparatus ofenhancing small or obscure populations for classification thereofdesignated generally by the reference numeral 700. This method andapparatus can be utilized to determine such small populations as the B'sor subsets of the L's. The analyzer 700 includes a biological sample702, which contains at least a first set of viable biological cells (notillustrated), such as in or from a whole blood sample. If theclassification is to be of B cells, then the sample 702 will include atleast the B cell population and at least one other WBC population, suchas the L cells. Generally the sample 702 would at least include all ofthe WBC populations, however, as previously described, variouspopulations or subsets thereof can be eliminated offline or in apre-preparation step. If the classification is to be of a WBC populationsubset, then the sample 702 will include at least one WBC populationhaving at least one definable subset.

Describing first the classification of B cells, the B cells are a verysmall population of the total WBC populations, generally on the order ofabout less than one percent. Clearly, eliminating the WBC populationswhich obscure the B cells, will enhance the analysis and classificationof the B's, because the B's then become a much greater and significantpart of the remaining WBC population, as the other WBC populations areeliminated. Clearly, also, this enhancement will be true for all smallor obscured cell populations of interest.

In analyzing the B cell populations utilizing a single measuringdimension or parameter, for example RF as illustrated in FIG. 38 to bedescribed hereinafter, the B's are obscured by the L's in a single peak704. The L's are around thirty (30) percent of the total WBC populationand hence the B's still are only about three (3) percent even of onlythe L's and B's together. Therefore, to more definitely analyze andclassify the B's, the L's are totally or substantially eliminated fromthe sample.

The sample 702 is combined via a line 706 with at least one reactant 708via a line 710. The RBC's are removed in an RBC removal station 712 byone of the methods previously described. Once the RBC's are removed, afirst portion of the resulting mixture is fed via a line 714 to a WBCanalyzer 716. The WBC analyzer can be the same as those previouslydescribed, or minor variations thereof. The single sensing parameter canbe electronic, such as RF or DC or light, such as Scatter or any otherdesired light parameter.

A second portion of the mixture is fed via a line 718 to a L removalstation 720, wherein the L cells are bound to magnetic microsphereswhich include an L specific monoclonal antibody or antibodies boundthereto. The remainder of the mixture with at least the B cellsremaining is fed via a line 722 to the WBC analyzer 716. The results ofthe two analyzed portions are fed to a comparator 724 via a line 726,where the two analyzed results are compared to determine the percentageof B's in the sample 702. The B's by this method have been made tochange from about one to three percent of the sample mixture tosubstantially one hundred percent in the first data peak 704, providingdefinitive analysis and characterization. In a like manner, when the CDor other WBC population group chosen is small, all or some of the restof the L's can be removed enhancing the analyzation of the remaining CDgroup of interest.

Next, describing the analysis and classification of one or more WBCpopulation subsets, for example, the CD2, CD4 or CD8 WBC populationsubsets. The sample 702 again will include at least a first set ofviable biological cells (not illustrated), such as in or from a wholeblood sample. The sample 702 will include at least the WBC populationsubset of interest and at least one other obscuring WBC population orpopulation subset and generally will include all the WBC populations.

The sample 702 again is fed to the RBC removal station 712 and then afirst portion is fed to the WBC analyzer 716 via the line 714. A secondportion will be utilized to determine the CD2 group, for example, andthus the second portion will be mixed with magnetic microspheres whichinclude a CD2 specific monoclonal antibody bound thereto, such as T11sold by Coulter Immunology Division of Coulter Corporation. Theremainder of the mixture, with the CD2 cells now removed, will be fedvia the line 722 to the WBC analyzer 716. The two analyzed results thenare compared in the comparator 724 to provide the desiredcharacterization of the CD2 group of cells.

The operation of the analyzer 700 can be performed on the instrument540, utilizing the respective channels as desired or on a furthermultichannel instrument or again on a single channel instrument in asequential fashion.

Referring now to FIGS. 38A-38E, the CD4, CD8 and CD2 subsets weredetermined as depicted in the one dimensional scattergramcharacterization results illustrated, utilizing a prototype analyzingmethod similar to the analyzer instrument 540. The biological sample ineach case was a 28 microliter sample of whole blood. The sample wascombined with 122 microliters of buffer solution for a control sampleutilized in the channel 714 (which can be the channel 514 of theinstrument 540). The results of a one dimensional electronic sensingparameter, here RF, was utilized to obtain the data for FIGS. 38A-E. Thesample portion was lysed for 4 seconds with 300 microliters of the RBCpreferential lyse above referenced (such as in the chamber 552) and thenquenched with 120 microliters of quench before being fed to the analyzer716 (such as the analyzer 558). This results in two clearly identifiabledata peaks 704 and 728 in FIG. 38A. Peak 704 includes the B's and L'sand all subsets thereof as a single peak. The B's are a small enoughpercentage so as to not effect the analyzation of the desired L subset,however, the value of the B's could be subtracted if desired.

A second control was utilized by combining a second whole blood sampleportion of 28 microliters with 50 microliters of magnetic microsphereswithout any L or L subset specific antibody bound thereto and 72microliters of buffer solution. The sample portion was lysed andquenched as before and then fed to the analyzer 716. This results in twodata peaks 730 and 732 in FIG. 38B. The data peaks 730 and 732 aresubstantially identical to the peaks 704 and 728, hence the inclusion ofthe microspheres did not appear to have any deleterious effects on theanalysis.

A third 28 microliter sample portion is fed to the channel 718 (whichcan be any one of the channels 518, 526 and 532 of the instrument 540),combined with 50 microliters of the magnetic microspheres with a CD4specific antibody bound thereto and 72 microliters of the buffersolution. The CD4 specific antibody can be T4 sold by Coulter ImmunologyDivision of Coulter Corporation. The sample portion was mixed for 60seconds, lysed and quenched as before and then placed in a magneticfield for 30 seconds, before the mixture with the CD4 subset populationremoved is fed to the analyzer 716. This results in two data peaks 734and 736 in FIG. 38C. The peak 734 represents the L's without the CD4subset population and then is compared to the peak 704 by the comparator724 to obtain the percentage of the CD4 subset population in the sample.

Again, for testing and evaluation purposes, the same blood sample wasanalyzed in the above manner to obtain four sets of data peaks, one ofwhich is actually depicted in FIGS. 38A-E. The data peaks in each set ofdata were substantially the same and hence a single set of data can beutilized. The data was averaged over the four sets of data to obtain theresults. The average CD4 subset population percentage obtained was 45.7,which was compared with a light sensing instrument for verificationpurposes, such as the EPICS@ flow cytometer available from CoulterCorporation, which provided a percentage of 47.6.

A fourth 28 microliter sample portion is fed to the channel 718,combined with 50 microliters of the magnetic microspheres with a CD8specific antibody bound thereto and 72 microliters of the buffersolution. The CD8 specific antibody can be T8 sold by Coulter ImmunologyDivision of Coulter Corporation. The sample portion was again mixed,lysed and quenched and placed in a magnetic field as before. The mixturewith the CD8 subset population removed then is fed to the analyzer 716.This results in two data peaks 738 and 740 in FIG. 38D. The data peak738 represents the L's without the CD8 subset population, which then iscompared to the peak 704 by the comparator 724 to obtain the percentageof the CD8 subset population in the sample. The average CD8 subsetpopulation percentage obtained was 25.0, which was compared with thelight sensing instrument for verification purposes, which provided apercentage of 26.0.

A fifth 28 microliter sample portion is fed to the channel 718, combinedwith 50 microliters of the magnetic microspheres with a CD2 specificantibody bound thereto and 72 microliters of the buffer solution. TheCD2 specific antibody can be T11 sold by Coulter Immunology Division ofCoulter Corporation. The sample portion was again mixed, lysed andquenched and placed in a magnetic field as before. The mixture with theCD2 subset population removed then is fed to the analyzer 716. Thisresults in two data peaks 742 and 744 in FIG. 38E. The data peak 742represents the L's without the CD2 subset population, which then iscompared to the peak 704 by the comparator 724 to obtain the percentageof the CD2 subset population in the sample. The average CD2 subsetpopulation percentage obtained was 82.7, which was compared with thelight sensing flow cytometer instrument, which provided a percentage of79.0.

The results of another subset analysis utilizing a one dimensionalelectronic sensing parameter of CD4, CD8 and CD2 subset populations isillustrated in FIGS. 39A-E and 40A-E. DC was utilized to obtain the datain FIGS. 39A-E, while RF was utilized to obtain the data in FIGS. 40A-E,utilizing the same sample portion and measured at the same time.

The sample portion was lysed and quenched before being fed to theanalyzer 716 in substantially the same manner as described with respectto FIGS. 38A-E. In the case of FIGS. 39A and 40A, only a buffer solutionwas added and in the case of FIGS. 39B and 40B, a buffer solution aswell as magnetic microspheres without the L or L subset specificantibodies were combined with the sample. The control withoutmicrospheres resulted in data peaks 746 and 748 in FIG. 39A and datapeaks 750 and 752 in FIG. 40A. Peaks 746 and 750 are representative ofthe L's and B's in the sample. The control with control microspherescombined with the sample, but with the microspheres removed therefromutilizing the magnetic field, resulted in data peaks 754 and 756 in FIG.39B and data peaks 758 and 760 in FIG. 40B.

A third whole blood sample portion is combined with 50 microliters ofmagnetic microspheres with a CD4 specific antibody bound thereto. Themixture is mixed, lysed, quenched and placed in the magnetic field toremove the CD4 subset population for the sample portion mixture beforeit is fed to the analyzer 716. The DC analysis results in two data peaks762 and 764 in FIG. 39C, while the RF analysis results in two data peaks766 and 768 in FIG. 40C. The data peaks 746 and 762 are compared as arethe data peaks 750 and 766 to obtain the percentage of the CD4 subsetpopulation in the sample.

In FIGS. 39 and 40, the data peaks are one of three separate sample setsfrom the same whole blood sample which were averaged to obtain theresults. In the case of CD4, the percentage obtained from DC was 57.0,RF was 57.1 and the light sensing flow cytometer instrument was 54.6.

To obtain the CD8 subset population analysis, a fourth whole bloodsample portion was combined with 50 microliters of magnetic microsphereswith a CD8 subset population specific antibody bound thereto. The sampleportion is mixed, lysed, quenched and held in a magnetic field to removethe CD8 subset population from the mixture, before the mixture is fed tothe analyzer 716. The DC analysis results in two data peaks 770 and 772in FIG. 39D, while the RF analysis results in two data peaks 774 and 776in FIG. 40D.

The data peaks 746 and 770 are compared as are the data peaks 750 and774 to obtain the percentage of the CD8 subset population in the sample.In the case of CD8, the percentage obtained from DC was 17.4, from RFwas 18.6 and from the light sensing flow cytometer instrument was 17.7.

To obtain the CD2 subset population analysis, a fifth whole blood sampleportion was combined with 50 microliters of magnetic microspheres with aCD2 subset population specific antibody bound thereto. The sampleportion is mixed, lysed, quenched and held in a magnetic field to removethe CD2 subset population from the mixture, before the mixture is fed tothe analyzer 716. The DC analysis results in two data peaks 778 and 780in FIG. 39E, while the RF analysis results in two data peaks 782 and 784in FIG. 40E.

The data peaks 746 and 778 are compared as are the data peaks 750 and782 to obtain the percentage of the CD8 subset population in the sample.In the case of CD8, the percentage obtained from DC was 79.6 from RF was79.3 and from the light sensing flow cytometer instrument was 74.7.

The B and L subset population analysis referred to with respect to FIGS.37-40 was obtained utilizing a one dimensional electronic sensingparameter. A light sensing parameter also could be utilized, as well astwo or more sensing parameters such as illustrated in FIGS. 41A-E.

The sample portions are handled in the same manner as those utilized toobtain the data in FIGS. 38-40. FIGS. 41A and 41B illustraterespectively, the results of a control without microspheres and acontrol combined with magnetic microspheres which are removed prior toanalyzing. The controls illustrate the normal three part histograms,illustrating the L's, M's and G's.

The CD4 depletion is illustrated in FIG. 41C, following the magneticdepletion of the CD4 subset population, the remaining L population canbe compared to the control L population to determine the CD4 subsetpopulation percentage. The CD4 subset population percentage wasdetermined to be 59.4, which was then compared to the light sensing flowcytometer instrument which determined a percentage of 54.6.

The CD8 depletion is illustrated in FIG. 41D, following the magneticdepletion of the CD8 subset population, the remaining L population canbe compared to the control L population to determine the CD8 subsetpopulation percentage. The CD8 subset population percentage wasdetermined to be 15.8, which was then compared to the light sensing flowcytometer instrument which determined a percentage of 17.7.

The CD2 depletion is illustrated in FIG. 41E, following the magneticdepletion of the CD2 subset population, the remaining L population canbe compared to the control L population to determine the CD2 subsetpopulation percentage. The CD2 subset population percentage wasdetermined to be 82.2, which was then compared to the light sensing flowcytometer instrument which determined a percentage of 74.7.

As can clearly be seen by the above analysis, a number of the subsetpopulations overlap, since the individual L subset populations add togreater than 100. "Overlapping" is utilized herein to signify thatcertain cells, populations of cells, subpopulations of cells or formedbodies include at least two receptors or antigens of interest.Overlapping can be a significant parameter in diagnosis and treatmentand will be discussed in further detail hereinafter.

All the data referred to heretofore, has been what would be called"normal" whole blood samples. "Normal" is utilized herein to signifythat a whole blood sample is not substantially infected, such as by acancer or other disease.

FIGS. 42 and 43 illustrate the results of analyzing two abnormal wholeblood samples. The first sample depicted in FIGS. 42A-G was analyzed anddisplayed in several different manners. FIGS. 42A and 42B depict twodifferent two dimensional sensing histograms of the whole blood sample,one with light sensing and one with only electronic sensing, without anytreatment of the blood sample. Clearly, the normal blood sample L, M andG grouping, for example, as illustrated in FIG. 41A is totally obscuredand there is just one unidentifiable data grouping.

The results of two different treatments of the abnormal whole bloodsample are illustrated in FIGS. 42C and 42D. In FIG. 42C, a 200microliter portion of the sample was combined with 200 microliters ofmagnetic microspheres having a N and E specific antibody bound thereto.The mixture is mixed, lysed, quenched and held in a magnetic field whilethe remaining portion is removed and fed to the analyzer without the Nand E bound cells therein. It appears clear that a substantial portionof the abnormal cells have been removed, since the pattern in FIG. 42Chas become very much more defined than the pattern in FIG. 42B.

In the second treatment, a 200 microliter portion of the sample wascombined with 200 microliters of magnetic microspheres having only a Nspecific antibody bound thereto. Although some of the cells have beenremoved, indicating that some are bound to the N antibody, a significantportion of the abnormal cells remain, especially as seen at the top ofthe histogram. It would therefore appear that some of the abnormal ordiseased cells bind to the N+E antibody. This type of depletiontreatment can be utilized for diagnosis and treatment of particulardiseases.

The data histograms of FIGS. 42A-D were developed utilizing twodimensional sensing. The same information can be developed utilizing asingle sensing parameter, for example, a single electronic sensingparameter as depicted in FIGS. 42E-42G. The one dimensional sensing,here DC, produces the histogram depicted in FIG. 42E when the sample isanalyzed without any treatment. The single data peak and the data at thefar right of the histogram are clear indications of an abnormal sample.

Again, the same treatment was utilized as referred to above with respectto FIGS. 42C and 42D and, in fact, the one dimensional sensing data wasgenerated at the same time as the two dimensional sensing data. Theanalyzing instrument can, as described above, include multiple sensingparameters or only one single parameter. Again, the removal of the N andE bound cells produces a histogram in FIG. 42F, which again illustratesthat most of the abnormal cells have been removed. The histogram in FIG.42G illustrates again that a significant number of the abnormal cellshave not been removed.

The results of analyzing the second abnormal whole blood sample areillustrated in FIGS. 43A-G. The results of analyzing the sample withouttreatment are illustrated in two dimensional histograms in FIGS. 43A and43B. Clearly, a normal whole blood sample data pattern is not seen. Thehistogram of FIG. 43B prepared in the instrument, can be compared to thehistogram in FIG. 43C prepared offline, which do not appearsignificantly different. A 28 microliter sample portion was preparedoffline or preprepared before the analysis depicted in FIG. 43C.

A portion of the sample was depleted of the CD5 subset population andthen analyzed to provide the histogram in FIG. 43D. A 28 microliterportion of the whole blood sample was combined with 122 microliters ofmagnetic microspheres having a CD5 specific antibody bound thereto, suchas T1 sold by Coulter Immunology Division of Coulter Corporation. Themixture was mixed, lysed, quenched and held in a magnetic field toremove the CD5 bound cells. This clearly removed a significant portionof the abnormal cells as can be seen by comparing FIGS. 43B or FIG. 43Cwith FIG. 43D. The histograms of FIGS. 43A-D were made with twodimensional sensing, while the same data is depicted in one dimensionhistograms in FIGS. 43E-G.

Again, no treatment was performed on the online sample depicted in FIG.43E or on the offline sample depicted in FIG. 43F and the CD5 subsetpopulation was depleted from the sample depicted in FIG. 43G.

As discussed hereinbefore, the overlapping populations of cells can beof interest for diagnostic as well as treatment of diseases of theblood. For example, some immature cells express both CD4 and CD8receptors. The obtaining of an analysis of the overlapping percentage ofcell subset populations has not been available when utilizing electronicsensing parameters or a minimal number of light parameters or simplifiedcombinations thereof. For example, in some light sensing instruments,three sensing parameters are utilized to obtain the overlapping data,both forward and 90° light scatter and fluorescence. One example ofoverlapping populations in a normal whole blood sample is the CD2 andCD8 subset populations. An abnormal overlapping of populations is foundin CLL (chronic lyphocytic leukemia). In the CLL disease state, the CD5and CD20 subset populations overlap.

Referring to FIG. 44, a first embodiment of a method and apparatus forperforming an overlapping classification of cells is designatedgenerally by the reference numeral 800. The instrument or analyzer 800includes a biological sample 802, which contains at least a first set ofviable biological cells (not illustrated), including at least twooverlapping white blood cell populations or subset populations, such asin or from a whole blood sample.

The cells of the biological sample 802 are to be involved in abiological reaction in a quantitative and/or qualitative determinationor analysis. The biological sample 802 can include a buffer into whichthe cells are added.

The biological sample 802 is combined via a line 804 with at least onereactant 806 via a line 808. In the analyzer 800, the RBC's are removedfrom the mixture at an RBC removing station 810. As stated in the firstparent application, the RBC's can be removed from the station 810 in anumber of ways, such as enumerated with respect to the station 20.

A first portion of the mixture with the RBC's removed, then is fed to aWBC analyzer 812 via a line 814. This obtains a standard or control forthe total or whole WBC populations of the biological sample 802. Theanalyzer 812 can be the same as the analyzer 86 or can be a lightsensing analyzer, such as described in U.S. Ser. No. 025,442 filed Mar.13, 1987 and U.S. Ser. No. 129,954, filed Dec. 4, 1987, entitledMULTI-PART DIFFERENTIAL ANALYZING APPARATUS UTILIZING LIGHT SCATTERTECHNIQUES, which are incorporated herein by reference. The singlesensing parameter can be electronic, such as RF or DC or light, such asmedian angle light scatter (Scatter) or any other desired lightparameter.

A second portion of the mixture is fed to an "X" removing station 816via a line 818. The station 816 removes a first overlapping populationof cells "X". The X's are removed or depleted by the addition of theappropriate magnetic microspheres with an X specific antibody boundthereto. A magnet or magnetic field is utilized, as before discussed, toremove the magnetically bound cells from the mixture. The remainingmixture with the X's removed then is fed via a line 820 to the analyzer812. The analyzed results of the "X" removed portion of the mixture thancan be compared with the analyzed results of the first mixture portionin a comparator 822 to obtain the percentage of X's in the biologicalsample 802.

A third portion of the mixture is fed to a second removing station 824via a line 826. The station 824 removes a second overlapping populationof cells "Y". The Y's are removed by the addition of appropriatemagnetic microspheres with a Y specific antibody bound thereto. Theremaining mixture with the Y's removed then is fed via a line 828 to theanalyzer 812. The analyzed results of the "Y" removed portion of themixture then is compared to the results of the first and second mixtureportions in the comparator 822 to verify if there is an overlapping ofthe X and Y populations in the biological sample 802. This operationverifies that the X and Y populations overlap, only if the X and Ypopulations or population ratios are generally known or if the total ofthe X and Y depleted populations is greater than 100%.

In most cases, a fourth portion of the mixture is fed to a removingstation 830 via a line 832. The station 830 removes both the X and Ypopulations (X+Y). The X's and Y's are removed by the addition of theappropriate magnetic microspheres with the X and Y specific antibodiesbound thereto. The remaining mixture with the X's and Y's removed thenis fed via a line 834 to the analyzer 812. The analyzed results of the"X"+"Y" removed portion of the mixture then can be compared to theresults of the other mixture portions to obtain the percentage ofoverlapping of the X and Y populations in the biological sample 802.

Thus, the analyzer 800 can perform a single overlapping classificationof cells, utilizing lines or channels 814, 818 and 826 and a fullpercentage overlapping classification utilizing all four lines. One mostimportant feature of the analyzer 800 is that the mixtures can beanalyzed utilizing only a single analyzing parameter, such as oneelectronic parameter or one light parameter. Other combinations can beutilized, but in each case only a single sensed parameter orcharacteristic is necessary to perform the overlapping classification ofthe parent application. The analysis also can be obtained with multiplesensing parameters.

A specific analyzing instrument embodiment incorporating the method andapparatus of the analyzer 800 is not illustrated, however, one suchinstrument can be the instrument 540. Again, the overlapping method andapparatus can be practiced on only a single channel of the instrument540 or on a single channel instrument, not illustrated.

Referring now to FIGS. 45A-45D, one set of one dimensional scattergramoverlapping characterization results are illustrated, obtained from awhole blood sample, utilizing a prototype analysis method similar to theanalyzer instrument 540 and described with respect to the instrument800. The biological sample in each case was a 28 microliter sample ofwhole blood, which was combined with 122 microliters of buffer solutionutilized in the first channel 814. The sample portion was lysed with 300microliters of the above referenced RBC preferential lyse for 4 secondsand then quenched with 120 microliters of quench before being fed to theanalyzer 812.

The data results of analyzing the portion with a one dimensionalelectronic sensing parameter, here DC, is illustrated in the histogramof FIG. 45A. The data results in two clearly identifiable data peaks 836and 838. As before, the peak 836 is indicative of the percentage of L'sand B's in the sample, while the peak 838 is indicative of thepercentage of N's, E's and M's.

The sample was then treated to deplete first the X and then the Y cellpopulations as above referenced. In this case, the X population was theCD2 subset population of the L's and the Y population was the CD20subset population of the L's. A second portion of the sample was fed tothe station 816, wherein 60 microliters of magnetic microspheres havinga CD2 specific antibody bound thereto was combined with the sampleportion, mixed, lysed, quenched and then held in a magnetic field toremove the CD2 subset population. The remaining mixture then was fed tothe analyzer 822 resulting in two data peaks 840 and 842 in FIG. 45B.The peak 840 is the remaining L's, which then is compared in thecomparator 822 to the peak 836 to obtain the percentage of the CD2subset population in the sample.

A third portion of the sample is fed to the station 824, wherein 50microliters of magnetic microspheres having a CD20 specific antibodybound thereto was combined with the sample portion, mixed, lysed,quenched and then held in a magnetic field to remove the CD20 subsetpopulation. The remaining CD20 removed mixture then was fed to theanalyzer 822 resulting in two data peaks 844 and 846 in FIG. 45C. Thepeak 844 is the remaining L's, which then is compared to the peak 836 toobtain the percentage of the CD20 subset population in the sample.

If the normal relative percentages of the CD2 and CD20 subsets areknown, then this can be sufficient to identify that the subsetpopulations are overlapping. Also, if the two subset populationpercentages add to a total of greater than 100 percent, then clearly,this also indicates that the two subset populations overlap. In cases ofsmall overlapping percentages, it then is important to obtain thepercentage of overlapping subset populations to verify that the subsetpopulations do overlap. A further depletion is necessary to obtain theoverlapping percentage.

A fourth portion of the sample is fed to the station 830, whereinmagnetic microspheres having the CD2 specific antibody and magneticmicrospheres having the CD20 specific antibody bound thereto arecombined to remove both the CD2 and CD20 subset populations. Theremaining CD2 and CD20 depleted mixture then was fed to analyzer 822resulting in two data peaks 848 and 850 in FIG. 45D. The peak 848 againis the remaining L's, which then is compared to the peak 836 to obtainthe percentages of subset populations removed by the CD20 and CD2specific antibodies. This result then is compared to the individual CD2and CD20 results to obtain the overlapping percentages, if any.

The exact overlapping percentages are calculated as follows:

I. Subset "A" (CD2) ##EQU2## II. Subset "B" (CD20) ##EQU3## III. Subset"A+B" (CD2+CD20) ##EQU4## IV. Overlapping Portion

    [Subset "A"+Subset "B"] -[(Subset "A+B"]=overlap

The results (averaged from two separate preparations) are illustrated inTable VI as follows:

                  TABLE VI                                                        ______________________________________                                                       Relative          Relative                                               Peak Percentage Peak   Percentage                                   ______________________________________                                        Control     836    35.7       838  64.3                                       CD2         840     7.1       842  92.9                                       CD20        844    33.6       846  66.4                                       CD2 + CD20  848     3.5       850  96.5                                       ______________________________________                                    

The relative percentages in Table VI are the percentage of the two peaksof the total percentage of 100. The percentage of CD2 was calculated as86.2, CD20 was 8.9 and CD2+CD20 was 93.5. Therefore, the overlappingpercentage was

    (CD2+CD20)-CD2+CD20 =(86.2+8.9)-93.5=1.6.

A second sample was depleted to obtain the percentage overlap of the CD2and CD8 subset populations as illustrated in FIGS. 46A-D. Again, a firstportion of the sample was fed to the channel 814 without a depletion toobtain the control data illustrated in FIG. 46A. The data results in twoclearly identifiable data peaks 852 and 854. The peak 852 again isindicative of the percentage of L's and B's in the sample.

A second sample portion is depleted of the CD2 subset population asbefore described in the channel 818, resulting in two data peaks 856 and858 in FIG. 46B. The remaining L peak 856 again is compared to thecontrol peak 852 to obtain the percentage of the CD2 subset populationin the sample.

A third sample portion is depleted of the CD8 subset population in thechannel 826, resulting in two data peaks 860 and 862 in FIG. 46C. Thepeak 860 is compared to the control peak 852 to obtain the percentage ofthe CD8 subset population in the sample.

A fourth sample portion is depleted of the CD2+CD8 subset populations inthe channel 832, resulting in two data peaks 864 and 866 in FIG. 46D.The peak 864 is compared to the control peak 852 to obtain thepercentage of CD2+CD8 subset populations in the sample.

The results are illustrated in Table VII as follows:

                  TABLE VII                                                       ______________________________________                                                      Relative           Relative                                              Peak Percentage  Peak   Percentage                                   ______________________________________                                        Control    852    30.3        854  69.7                                       CD2        856     9.0        858  91.0                                       CD8        860    22.1        862  77.9                                       CD2 + CD8  864     7.6        866  92.4                                       ______________________________________                                    

The percentage of CD2 was calculated as 77.3, CD8 was 34.7 and CD2+CD8was 81.1. Therefore, the overlapping percentage was(77.3+34.7)-81.1=30.9.

Referring now to FIGS. 47-60, the embodiments of the present inventionare illustrated.

Referring to FIG. 47, a first embodiment of a method and apparatus forperforming classifications of obscured or partially obscured cells,specifically, for example purposes those cells forming a white bloodcell population subset of interest is designated generally by thereference numeral 900. The instrument or analyzer 900 includes abiological sample 902, which contains at least a first set of viablebiological cells (not illustrated), including at least two white bloodcell populations, such as in or from a whole blood sample. At least oneof the white blood cell populations includes a white blood cellpopulation subset of interest, the sensed characteristics of which areobscured by the white blood cell population. The sensed characteristicsare to be shifted by binding microspheres having monoclonal antibodiesspecific thereto to the white blood cell population subset, however, theshifted sensed characteristic will be obscured or partially obscured bythe second white blood cell population.

The cells of the biological sample 902 are to be involved in abiological reaction in a quantitative and/or qualitative determinationor analysis. The biological sample 902 can include a buffer into whichthe cells are added. The biological sample 902 is combined via a line904 with at least one reactant 906 via a line 908. In the analyzer 900,the RBCs are removed from the mixture at an RBC removing station 910.

As stated in the first parent application, the RBC's can be removed fromthe station 910 in a number of ways, utilizing lyse and/or microspheres,such as enumerated with respect to the station 20.

A first portion of the mixture with the RBC's removed, then is fed to awhite blood cell analyzer 912 via a line 914. This obtains a standard orcontrol for analyzing the white blood cell populations of the biologicalsample 902 for an obscured white blood cell subset population ofinterest. The standard population can be one of the total number ofwhite blood cell populations, a second white blood cell population whichdoes not obscure the shifted or non-shifted sensed characteristic of thesubset of interest, an artificial population formed by microsphereswhich also do not obscure the shifted or non-shifted sensedcharacteristic of the subset of interest or a white blood cellpopulation into which the sensed characteristic of the subset will bewholly or partially shifted. The artificial population can be formedfrom microspheres which do not have antibodies or cells bound theretoand form a sensed characteristic which does not obscure the first whiteblood cell population or the shifted characteristic of the white bloodcell subset of interest. The analyzer 912 can be the same as theanalyzer 86. The analyzer 912 utilizes two electronic sensingparameters, such as RF or DC.

The white blood cell population subset of interest is obscured in one ofthe sensed white blood cell populations. A second portion of the mixtureis fed to a white blood cell population subset shifting station 916 viaa line 918. The white blood cell population subset is bound to whiteblood cell microspheres having monoclonal antibodies specific to thesubset thereon to modify (change or shift) the resultant opacity and/orvolume parameters of the cells.

The mixture with the white blood cell population subset shifted, then isfed to the analyzer 912 via a line 920. The white blood cell populationsubset of interest generally is related as a percentage of the whiteblood cell population of interest. The results of the white blood cellanalyzer 912 from the original or non-shifted cell mixture then iscompared in a comparator 922 to obtain the percentages of the whiteblood cell population.

The instrument 900 can be substantially identical to the instrument 540,utilizing only the lines 514 and 518, without the magnetic field 592.The instrument 900 also can be a sequential type instrument, such as theinstrument 56, in which the same sample portion can be counted, thenshifted, recounted and compared.

Referring to FIG. 48A, a conceptual or idealized scattergram isillustrated which shows the conventional scattergram results of thesensed characteristics of at least two groups or populations 926 and 928of cells or formed bodies. The population 926 includes an obscuredpopulation or subset of cells 927 which is obscured or masked by thepopulation 926 and cannot be identified separately therefrom.Microspheres having reacting agents bound thereto which are specific toor detect specific molecules on the cells of the population 927 arecombined therewith, however the sensed characteristics of the 927 cellsbound with or to the reacting agent microspheres will be shifted fromthe 926 population and be wholly or partially obscured by the population928 forming a population 927', as illustrated in FIG. 48B. Forillustration and example purposes, utilizing WBC's, a population 924 isM's, the population 926 is L's, the populations 927 and 927' are Lsubsets and the population 928 is G's. For example purposes, thescattergram results are shown as discrete areas, although the areas 924,926 and 928 are actually a plurality of individual sensed data points.Also, the scattergram is depicted utilizing RF and opacity, howeverother parameters can be utilized such as DC or other combinations asgenerally shown in FIGS. 26A-D and FIGS. 57-60. In addition to theconventional sensed data groups 924, 926 and 928, a fourth artificialpopulation 930 is illustrated, such as a population of microsphereswithout cells or antibodies bound thereto, which also can be utilized inthe present invention.

In the present invention, the standard or control population includessensing and counting at least the L's 926 and various combinations ofthe other groups to determine the white blood cell subset of interest,which in the examples are L subsets such as CD4 or CD8. The standardpopulation is obtained, because the sensed characteristics of the Lsubset of interest is shifted into the G population 928 which wholly orpartially obscures the L subset of interest. Generally, to obtain acorrect analysis of the percentage contribution of the subset populationof interest, a control population has to be obtained before and afterthe shift, to normalize the subset percentage.

As above described, the standard population can be:

(1) the total number of white blood cell populations 924, 926 and 928,in which case the artificial population 930 generally would not beutilized;

(2) the M population 924 which does not obscure the shifted ornon-shifted sensed characteristics of the subset of interest;

(3) the artificial population 930 which also does not obscure theshifted or non-shifted sensed characteristics of the subset of interest;or

(4) the G population 928 into which the sensed characteristics of thesubset will be wholly or partially shifted. The standard populationprovides the normalization population for the methodology of the presentinvention.

Referring now to FIGS. 49A and 49B, two sets of results depicted inscattergrams obtained from a whole blood sample utilizing a method inaccordance with the instrument 900, are illustrated. Referring to FIG.49A, a non-shifted or control scattergram is depicted. In this example,the CD8 subset of the lymphocytes is the white blood cell populationsubset of interest.

The control biological sample 902 is a 25 microliter sample of wholeblood combined with 100 microliters of buffer. The reactant 906 is 300microliters of RBC lyse, previously referred to, which is combined withthe biological sample 902 and mixed for five seconds. The mixture isquenched with 120 microliters of quench, previously referred to, whichis mixed again for five seconds and fed to the white blood cell analyzer912. The white blood cell analyzer 912 obtains a first lymphocyte countL1 of 965, which includes the CD8 white blood cell population subset ofinterest. The lymphocytes are contained in a block 932, which preferablyis gated at 934 to eliminate debris, such as aged neutrophils from thecount. A first total white blood cell count T1 of 4862 is also obtained,which includes the cells in block 932 as well as the cells in M and Gcell groups 936 and 938 outside the block 932.

The sample mixture 902 or a second portion thereof then is mixed for 15seconds with 50 microliters of 2.2 micron microspheres in a 2 percentsolution with a T8 specific monoclonal antibody bound thereto. If asecond portion of the mixture 902 is utilized, the RBC's would beeliminated by one of the above referenced methods. The shifted mixturethen is fed to the white blood cell analyzer 912 to obtain thescattergram depicted in FIG. 49B. The CD8 white blood cell populationsubset now has been shifted from the lymphocyte cells in the block 932'into the G group of cells 938'.

The CD8 white blood cell population subset was obscured or partiallyobscured by the L's in block 932 and now are obscured or partiallyobscured by the G cells in the cell group 938'. The white blood cellanalyzer 912 obtains a second count L2 of L's of 764 (reduced from 965)and a second total white blood cell count T2 of 4864.

If the total white blood cell counts were identical, then the CD8 countcould be obtained merely by subtraction. However, if this is not thesituation, and generally it would not be, then the percentage can benormalized, for example, by utilizing the two total white blood cellcounts as follows: ##EQU5## The CD8 percentage obtained was compared tothe percentage obtained by flow cytometry, which was 23.0%. Othercontrol populations can be utilized as previously described.

The CD4 white blood cell population subset percentage can be obtained ina similar manner as illustrated in FIGS. 50A and 50B. In this examplethe microspheres are bound to T4 specific monoclonal antibodies and aremixed for 30 seconds. The control scattergram is depicted in FIG. 50A.Again, a first lymphocyte count 1171 of the L's in a block 940 isobtained and a first total white blood cell count of 4863 is obtained,including the groups of M and G cells 942 and 944.

The shifted scattergram is depicted in FIG. 50B, which provides a secondL count of 878 and a second white blood cell total count of 4857. Thetotal CD4 white blood cell subset population is 24.9 percent, whichcompares to a flow cytometry count of 28.6 percent.

In the above examples, the scattergrams were obtained by utilizing RFand opacity. Other sensing parameters also can be utilized, see forexample FIGS. 57-60, the RF parameter however provides a gooddemarcation between the L's and the remaining white blood cells. Also,all the white blood cells are counted in the examples, but the M's ingroup 936 and 942 can be ignored, since the M's do not obscure eitherthe shifted or unshifted CD4 or CD8 white blood cell population subsetof interest.

Three additional examples of CD8 percentages from different samples aredepicted in FIGS. 51-53. In FIG. 51A, a first L count of 1520 wasobtained in a block 946, while a first total white blood cell count of4861 was obtained counting the L's in the block 946 and the remainingcells and cell groups in a block 948. The second shifted L count of 1030was obtained in a block 946' of FIG. 51B, while the second total whiteblood cell count of 4856 was obtained including the cells in blocks 946'and 948'. The CD8 percentage obtained was 32.2 while flow cytometryprovided a percentage of 34.6.

In the same manner, the scattergrams of FIGS. 52A and 52B provided firstand second L counts of 1393 and 930 and first and second white bloodcell counts of 9864 and 4861. The CD8 percentage obtained was 33.2,which compared to the flow cytometry percentage of 31.3. Thescattergrams of FIGS. 53A and 53B provided first and second L counts of1031 and 778 and first and second white blood cell counts of 4862 and4860. The CD8 percentage obtained was 24.5, which compared to the flowcytometry percentage of 21.7.

Three additional examples of the CD4 percentage obtained from differentsamples are depicted in FIGS. 54-56. In FIG. 54A, a first L count of1372 was obtained in a block 950, while a first total white blood cellcount of 4764 was obtained counting the L's in the block 950 and theremaining cells and cell groups in a block 952. The second shifted Lcount of 670 was obtained in the block 950' in FIG. 54B, while thesecond total white blood cell count of 4855 was obtained including thecells in blocks 950' and 952'. The CD4 percentage obtained was 52.1,while the flow cytometry provided a percentage of 54.4.

In the same manner, the scattergrams of FIGS. 55A and 55B, providedfirst and second L counts of 1684 and 956 and first and second whiteblood cell counts of 4863 and 4862. The CD4 percentage obtained was43.2, which compared to the flow cytometry percentage of 45.3. Thescattergrams of FIGS. 56A and 56B provided first and second L counts of1234 and 542 and first and second white blood cell counts of 4859 and4857. The CD4 percentage obtained was 56.1, which compared to the flowcytometry percentage of 56.3.

FIGS. 57-60 illustrate other combinations of electronic sensingparameters to form the scattergrams. In FIGS. 57A and B, the scattergramutilizes DC and opacity for the illustration. A first L count of 868 wasobtained in a block 954, while a first total white blood cell count of4827 was obtained counting the L's in the block 954 and the remainingcells and cell groups in a block 956. The second shifted L count of 664was obtained in a block 954' of FIG. 57B, while the second total whiteblood cell count of 4813 was obtained including the cells in blocks 954'and 956'. The CD8 percentage obtained was 23.2.

The scattergrams of FIGS. 58A and 58B are illustrated utilizing DC andRF as the sensing parameters. In FIG. 58A, a first L count of 926 wasobtained in a block 958, while a first total white blood cell count of4848 was obtained counting the L's in the block 958 and the remainingcells and cell groups in a block 960. The second shifted L count of 776was obtained in a block 958' of FIG. 58B, while the second total whiteblood cell count of 4855 was obtained including the cells in blocks 958'and 960'. The CD8 percentage obtained was 16.3.

The scattergrams of FIGS. 59A and 59B are illustrated utilizing only asingle, DC, sensing parameter. In FIG. 59A a first L count of 935 wasobtained in a block 962, while a first total white blood cell count of3918 was obtained counting the L's in the block 962 and the remainingcells and cell groups in a block 964. The second shifted L count of 825was obtained in a block 962' of FIG. 59B, while the second total whiteblood cell count of 4029 was obtained including the cells in blocks 962'and 964'. The CD8 percentage obtained was 11.8.

The scattergram of FIGS. 60A and 60B are illustrated utilizing only asingle, RF, sensing parameter. In FIG. 60A a first L count of 969 wasobtained in a block 966, while a first total white blood cell count of3880 was obtained counting the L's in the block 966 and the remainingcells and cell groups in a block 968. The second shifted L count of 779was obtained in a block 966' of FIG. 60B, while the second total whiteblood cell count of 4069 was obtained including the cells in blocks 966'and 968'. The CD8 percentage obtained was 19.6.

The scattergrams of FIGS. 57-60 are not optimized and the data asdepicted is not clearly separated. This can be seen especially from theone-dimensional sensing of FIGS. 59A and B and FIGS. 60A and B sinceeach of the Figures are derived from the same data. This is especiallypronounced in FIGS. 60A and B, since a result was obtained of 11.8,which is far from the result of 23.2 obtained in FIGS. 57A and B. Also,although 2.2 diameter micron microspheres were utilized for examplepurposes, other sizes also can be utilized.

In the above described examples, the standard population utilized is thetotal count of the total white blood cell populations, since the totalcount should remain constant. The standard population also can be thenon-interfering white blood count 924 or artificial population 930 (FIG.48), which is unaffected or non-obscured by the white blood populationsubset of interest, before or after it is shifted. Since the white bloodcell population is unaffected, the first and second counts thereofshould be the same except for cell fragments, errant microspheres orother aberrations. The artificial population can be formed by a set ofmicrospheres, which also are unobscured.

Also, although the method and the apparatus of the present inventionhave been described utilizing whole blood samples, there can beinstances where it is desired to utilize a portion of a sample with theRBC's and/or some of the WBC populations removed. Clearly, the RBC's arestill removed, but arguably externally and not within the apparatus ofthe present invention. Such removal or prepreparation can be carried outin numerous conventional ways, such as utilizing a lysing reagent,density or centrifugation techniques, such as ficoll, dextran,"buffycoat", etc. In an automated analyzer utilizing the presentinvention, it would be preferable to utilize a whole blood sample forspeed and integrity in the analysis of the sample.

Many modifications and variations of the present invention are possiblein light of the above teachings. The samples 12, 42, 150, 180, 294, 322,342 and 902 can include whole blood, human body fluids containing cells,or other fluids containing formed bodies, such as bacteria, viruses andfungi. The volumes of microspheres specified are stated in weight ofmicrospheres per volume of diluent. Although volumes on the order ofabout 20 microliters of sample, such as a whole blood sample, have beenutilized for example purposes herein, smaller or larger sample volumesalso can be utilized as desired. For example, as small as about 2microliters of a sample up to whatever volume of sample is practical forthe particular instrument or technique can be utilized. Although some ofthe examples were performed in sequential steps, the steps can also beperformed simultaneously. A simultaneous analysis allows the leastcomplex instrument module to be utilized. It is therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

What is claimed and desired to be secured by Letters Patent of theUnited States is:
 1. A method of obtaining at least one obscured orpartially obscured population analysis from at least a portion of asample having at least a first cell population including at least onepopulation subset of interest and a second cell population,comprising:electronically sensing and counting a first populationincluding at least the first cell population and subsets thereof to forma first count; shifting the cell population subset of interest out ofsaid first cell population and at least partially into a second cellpopulation by binding microspheres having a reactant bonded theretospecific to said cell population subset of interest to said cellpopulation subset; electronically sensing and counting the remainingfirst population including at least the first cell population withoutthe shifted subset and with remaining subsets thereof to form a secondcount; and comparing said first and second counts to obtain thepercentage contribution of the cell population subset of interest. 2.The method as defined in claim 1 including electronically sensing andcounting said populations utilizing two different electronic parameters.3. The method as defined in claim 1 including electronically sensing andcounting said populations utilizing a single electronic parameter. 4.The method as defined in claim 1 including shifting said cell populationsubset of interest by mixing said microspheres with said sample to bindto said cell population subset of interest and shift at least one sensedcharacteristic of said cell population subset of interest.
 5. The methodas defined in claim 4 including providing monoclonal antibodies formingsaid reactants with antigens on said cell population subset of interestforming said specific molecule.
 6. The method as defined in claim 1including providing a whole blood sample having at least a first whiteblood cell population forming said first cell population and a secondwhite blood cell population forming said second cell population.
 7. Themethod as defined in claim 6 including said first white blood cellpopulation including a CD4 white blood cell Dopulation subset andshifting the CD4 white blood cell population subset by providingmicrospheres having a monoclonal antibody bonded thereto which isspecific to said CD4 white blood cell population subset and mixing saidmicrospheres with said sample to bind to said CD4 subset population toshift at least one sensed characteristic of said CD4 subset population.8. The method as defined in claim 6 including said first white bloodcell population including a CD8 white blood cell population subset andshifting the CD8 white blood cell population subset by providingmicrospheres having a monoclonal antibody bonded thereto which isspecific to said CD8 white blood cell population subset and mixing saidmicrospheres with said sample to bind to said CD8 subset population toshift at least one sensed characteristic of said CD8 subset population.9. The method as defined in claim 6 wherein said whole blood sampleincludes a red blood cell population and including removing said redblood cell population from said sample without significantly adverselyaffecting relevant qualities and/or quantities of said white blood cellpopulations.
 10. The method as defined in claim 6 including sensing andcounting at least the first white blood cell population and subsetsthereof and the second white blood cell population to form a third countprior to said shifting of the white blood cell population subset ofinterest and sensing and counting at least the remaining first whiteblood cell population and subsets thereof and the second white bloodcell population including said shifted white blood cell subset ofinterest to form a fourth count following said shifting of the whiteblood cell population subset of interest and comparing said first andsecond counts to at least one of said third and fourth counts to obtainthe percentage contribution of the white blood cell population subset ofinterest, said microspheres being of a smaller size than said cellssufficient to shift cells to an area on a scattergram where they can beseparately identified and counted from the other cells which have notbeen shifted.
 11. The method as defined in claim 10 including comparingsaid first and second counts to both of said third and fourth counts tonormalize the percentage contribution.
 12. The method as defined inclaim 10 including sensing and counting all of the white blood cellpopulations and subsets thereof to form said third and fourth counts.13. The method as defined in claim 6 including sensing and counting astandard population to form a third count prior to said shifting of thewhite blood cell population subset of interest and sensing and countingsaid standard population following said shifting of the white blood cellpopulation subset of interest to form a fourth count and comparing saidthird and fourth counts to normalize the percentage contribution. 14.The method as defined in claim 13 wherein said standard population is anartificial population formed by non-interfering microspheres.
 15. Themethod as defined in claim 13 where said standard population is anon-interfering white blood cell population.
 16. The method as definedin claim 13 wherein said microspheres are of a smaller size than saidcells sufficient to shift cells to an area on a scattergram where theycan be separatelv identified and counted from the other cells which havenot been shifted, and said standard population is a total of all thewhite blood cell populations.
 17. An apparatus for obtaining at leastone obscured or partially obscured population analysis from at least aportion of a sample having at least a first cell population including atleast one population subset of interest and a second cell populationcomprising:means for electronically sensing and counting a firstpopulation including at least the first cell population and subsetsthereof to form a first count; means for shifting the cell populationsubset of interest out of said first cell population and at leastpartially into a second cell population by binding microspheres having areactant bonded thereto specific to said cell population subset ofinterest to said cell population subset; means for electronicallysensing and counting the remaining first population including at leastthe first cell population without the shifted subset and with remainingsubsets thereof to form a second count; and means for comparing saidfirst and second counts to obtain the percentage contribution of thecell population subset of interest.
 18. The apparatus as defined inclaim 17 including means for electronically sensing and counting saidpopulations utilizing two different electronic parameters.
 19. Theapparatus as defined in claim 17 including means for electronicallysensing and counting said populations utilizing a single electronicparameter.
 20. The apparatus as defined in claim 17 including means forshifting said cell population subset of interest by mixing saidmicrospheres with said sample to bind to said cell population subset ofinterest and shift at least one sensed characteristic of said cellpopulation subset of interest.
 21. The apparatus as defined in claim 20including means for providing monoclonal antibodies forming saidreactants with antigens on said cell population subset of interestforming said specific molecule.
 22. The apparatus as defined in claim 17including a whole blood sample having at least a first white blood cellpopulation forming said first cell population and a second white bloodcell population forming said second cell population.
 23. The apparatusas defined in claim 22 including said first white blood cell populationincluding a CD4 white blood cell population subset and means forshifting the CD4 white blood cell population subset including means forproviding microspheres having a monoclonal antibody bonded thereto whichis specific to said CD4 white blood cell population subset and means formixing said microspheres with said sample to bind to said CD4 subsetpopulation to shift at least one sensed characteristic of said CD4subset population.
 24. The apparatus as defined in claim 22 said firstwhite blood cell population including a CD8 white blood cell populationsubset and including means for shifting the CD8 white blood cellpopulation subset including means for providing microspheres having amonoclonal antibody bonded thereto which is specific to said CD8 whiteblood cell population subset and means for mixing said microspheres withsaid sample to bind to said CD8 subset population to shift at least onesensed characteristic of said CD8 subset population.
 25. The apparatusas defined in claim 22 wherein said whole blood sample includes a redblood cell population and including means for removing said red bloodcell population from said sample without significantly adverselyaffecting relevant qualities and/or quantities of said white blood cellpopulations.
 26. The apparatus as defined in claim 22 including meansfor sensing and counting at least the first white blood cell populationand subsets thereof and the second white blood cell population to form athird count prior to said shifting of the white blood cell populationsubset of interest and means for sensing and counting at least theremaining first white blood cell population and subsets thereof and thesecond white blood cell population including said shifted white bloodcell subset of interest to form a fourth count following said shiftingof the white blood cell population subset of interest and means forcomparing said first and second counts to at least one of said third andfourth counts to obtain the percentage contribution of the white bloodcell population subset of interest, said microspheres being of a smallersize than said cells sufficient to shift cells to an area on ascattergram where they can be separately identified and counted from theother cells which have not been shifted.
 27. The apparatus as defined inclaim 26 including means for comparing said first and second counts toboth of said third and fourth counts to normalize the percentagecontribution.
 28. The apparatus as defined in claim 26 including meansfor sensing and counting all of the white blood cell populations andsubsets thereof to form said third and fourth counts.
 29. The apparatusas defined in claim 22 including means for sensing and counting astandard population to form a third count prior to said shifting of thewhite blood cell population subset of interest and means for sensing andcounting said standard population following said shifting of the whiteblood cell population subset of interest to form a fourth count andmeans for comparing said third and fourth counts to normalize thepercentage contribution.
 30. The apparatus as defined in claim 29wherein said standard population is an artificial population formed bynon-interfering microspheres.
 31. The apparatus as defined in claim 29where said standard population is a non-interfering white blood cellpopulation.
 32. The apparatus as defined in claim 29 wherein saidmicrospheres are of a smaller size than said cells sufficient to shiftcells to an area on a scattergram where they can be separatelyidentified and counted from the other cells which have not been shifted,and said standard population is a total of all the white blood cellpopulations.